Use of thiol redox proteins for reducing protein intramolecular disulfide bonds, for improving the quality of cereal products, dough and baked goods and for inactivating snake, bee and scorpion toxins

ABSTRACT

Methods of reducing cystine containing animal and plant proteins, and improving dough and baked goods&#39; characteristics is provided which includes the steps of mixing dough ingredients with a thiol redox protein to form a dough and baking the dough to form a baked good. The method of the present invention preferably uses reduced thioredoxin with wheat flour which imparts a stronger dough and higher loaf volumes. Methods for reducing snake, bee and scorpion toxin proteins with a thiol redox (SH) agent and thereby inactivating the protein or detoxifying the protein in an individual are also provided. Protease inhibitors, including the Kunitz and Bowman-Birk trypsin inhibitors of soybean, were also reduced by the NADP/thioredoxin system (NADPH, thioredoxin, and NADP-thioredoxin reductase) from either  E. coli  or wheat germ. When reduced by thioredoxin, the Kunitz and Bowman-Birk soybean trypsin inhibitors lose their ability to inhibit trypsin. Moreover, the reduced form of the inhibitors showed increased susceptibility to heat and proteolysis by either subtilisin or a protease preparation from germinating wheat seeds. The 2S albumin of castor seed endosperm was reduced by thioredoxin from either wheat germ or  E. coli . Thioredoxin was reduced by either NADPH and NADP-thioredoxin reductase or dithiothreitol. Analyses showed that thioredoxin actively reduced the intramolecular disulfides of the 2S large subunit, but was ineffective in reducing the intermolecular disulfides that connect the large to the small subunit. A novel cystine containing protein that inhibits pullulanase was isolated. The protein was reduced by thioredoxin and upon reduction its inhibitory activity was destroyed or greatly reduced.

CROSS-REFERENCE

This application is a divisional application of application of Ser. No.08/211,673, filed Nov. 21, 1994 (now U.S. Pat. No. 6,113,951 issued Sep.5, 2000), which is a national stage filing of International ApplicationNo. PCT/US92/08595 filed Oct. 8, 1992, which is a continuation-in-partof application Ser. No. 07/935,002, filed Aug. 25, 1992 (now abandoned),which is a continuation-in-part application of Ser. No. 07/776,109, Oct.12, 1991 (now abandoned).

Reference is also made to the following related applications andpatents: (1) application Ser. No. 08/326,976, filed Oct. 21, 1994 (nowU.S. Pat. No. 5,792,506 issued Aug. 11, 1998), which is acontinuation-in-part of application Ser. No. 08/211,673 filed Nov. 21,1994 (now U.S. Pat. No. 6,113,951 issued Sep. 5, 2000); (2) applicationSer. No. 08/483,390, filed Jun. 7, 1995 (now U.S. Pat. No. 6,114,504issued Sep. 5, 2000), which is a divisional of application Ser. No.08/211,673 (now U.S. Pat. No. 6,113,951 issued Sep. 15, 2000); (3)application Ser. No. 08/953,073, filed Oct. 17, 1997 (now U.S. Pat. No.5,952,034 issued Sep. 14, 1999), which is a continuation-in-part ofapplication Ser. No. 08/326,976 (now U.S. Pat. No. 5,792,506, issuedAug. 11, 1998); (4) application Ser. No. 09/046,780 filed Mar. 23, 1998(now U.S. Pat. No. 6,190,723 (issued Feb. 20, 2001), which is adivisional of application Ser. No. 08/326,976, U.S. Pat. No. 5,792,506);(5) application Ser. No. 09/238,379 filed Jan. 27, 1999 now abandoned,which is a continuation-in-part of application Ser. No. 08/953,703 (nowU.S. Pat. No. 5,952,034 issued Sep. 14, 1999); (6) application Ser. No.09/296,927 filed Mar. 22, 1999 now abandoned, which is a divisional ofapplication Ser. No. 08/953,703 (U.S. Pat. No. 5,952,034); (7)application Ser. No. 09/447,615 filed Nov. 23, 1999, which is adivisional of application Ser. No 08/211,673 (U.S. Pat. No. 6,113,951);and (8) application Ser. No. 09/448,111 filed Nov. 23, 1999, which is adivisional of application Ser. No. 08/211,673 U.S. Pat. No. 6,113,951.

FIELD OF THE INVENTION

The present invention relates to the use of thiol redox proteins toreduce seed protein such as cereal proteins, enzyme inhibitor proteins,venom toxin proteins and the intramolecular disulfide bonds of certainother proteins. More particularly, the invention involves use ofthioredoxin and glutaredoxin to reduce gliadins, glutenins, albumins andglobulins to improve the characteristics of dough and baked goods andcreate new doughs and to reduce cystine containing proteins such asamylase and trypsin inhibitors so as to improve the quality of feed andcereal products. Additionally, the invention involves the isolation of anovel protein that inhibits pullulanase and the reduction of that novelprotein by thiol redox proteins. The invention further involves thereduction by thioredoxin of 2S albumin proteins characteristic ofoil-storing seeds. Also, in particularly the invention involves the useof reduced thiol redox agents to inactivate snake neurotoxins andcertain insect and scorpion venom toxins in vitro and to treat thecorresponding toxicities in individuals.

This invention was made with government support under Grant ContractNos. DCB 8825980 and DMB 88-15980 awarded by the National ScienceFoundation. The United States Government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

Chloroplasts contain a ferredoxin/thioredoxin system comprised offerredoxin, ferredoxin-thioredoxin reductase and thioredoxins f and mthat links light to the regulation of enzymes of photosynthesis(Buchanan, B. B. (1991) “Regulation of CO₂ assimilation in oxygenicphotosynthesis: The ferredoxin/thioredoxin system. Perspective on itsdiscovery, present status and future development”, Arch. Biochem.Biophys. 288:1-9; Scheibe, R. (1991), “Redox-modulation of chloroplastenzymes. A common principle for individual control”, Plant Physiol.96:1-3). Several studies have shown that plants also contain a system,analogous to the one established for animals and most microorganisms, inwhich thioredoxin (h-type) is reduced by NADPH and the enzyme,NADP-thioredoxin reductase (NTR) according to the following:

(Florencio F. J., et al. (1988), Arch. Biochem. Biophys. 266:496-507;Johnson, T. C., et al. (1987), Plant Physiol. 85:446-451; Suske, G., etal. (1979), Z. Naturforsch. C. 34:214-221). Current evidence suggeststhat the NADP/thioredoxin system is widely distributed in plant tissuesand is housed in the mitochondria, endoplasmic reticulum and cytosol(Bodenstein-Lang, J., et al. (1989), FEBS Lett. 258:22-26; Marcus, F.,et al. (1991), Arch. Biochem. Biophys. 287:195-198).

Thioredoxin h is also known to reductively activate cytosolic enzyme ofcarbohydrate metabolism, pyrophosphate fructose-6-P,1-phosphotransferase or PFP (Kiss, F., et al. (1991), Arch. Biochem.Biophys. 287:337-340).

The seed is the only tissue for which the NADP/thioredoxin system hasbeen ascribed physiological activity in plants. Also, thioredoxin h hasbeen shown to reduce thionins in the laboratory (Johnson, T. C., et al.(1987), Plant Physiol. 85:446-451). Thionins are soluble cereal seedproteins, rich in cystine. In the Johnson, et al. investigation, wheatpurothionin was experimentally reduced by NADPH via NADP-thioredoxinreductase (NTR) and thioredoxin h according to Eqs. 2 and 3.

Cereal seeds such as wheat, rye, barley, corn, millet, sorghum and ricecontain four major seed protein groups. These four groups are thealbumins, globulins, gliadins and the glutenins or correspondingproteins. The thionins belong to the albumin group or faction.Presently, wheat and rye are the only two cereals from which gluten ordough has been formed. Gluten is a tenacious elastic and rubbery proteincomplex that gives cohesiveness to dough. Gluten is composed mostly ofthe gliadin and glutenin proteins. It is formed when rye or wheat doughis washed with water. It is the gluten that gives bread dough itselastic type quality. Flour from other major crop cereals barley, corn,sorghum, oat, millet and rice and also from the plant, soybean do notyield a gluten-like network under the conditions used for wheat and rye.

Glutenins and gliadins are cystine containing seed storage proteins andare insoluble. Storage proteins are proteins in the seed which arebroken down during germination and used by the germinating seedling togrow and develop. Prolamines are the storage proteins in grains otherthan wheat that correspond to gliadins while the glutelins are thestorage proteins in grains other than wheat that correspond toglutenins. The wheat storage proteins account for up to 80% of the totalseed protein (Kasarda, D. D., et al. (1976), Adv. Cer. Sci. Tech.1:158-236; and Osborne, T. B., et al. (1893), Amer. Chem. J.15:392-471). Glutenins and gliadins are considered important in theformation of dough and therefore the quality of bread. It has been shownfrom in vitro experiments that the solubility of seed storage proteinsis increased on reduction (Shewry, P. R., et al. (1985), Adv. Cer. Sci.Tech. 7:1-83). However, previously, reduction of glutenins and gliadinswas thought to lower dough quality rather than to improve it (Dahle, L.K., et al. (1966), Cereal Chem. 43:682-688). This is probably becausethe non-specific reduction with chemical reducing agents caused theweakening of the dough.

The “Straight Dough” and the “Pre-Ferment” methods are two majorconventional methods for the manufacture of dough and subsequent yeastraised bread products.

For the Straight Dough method, all of the flour, water or other liquid,and other dough ingredients which may include, but are not limited toyeast, grains, salt, shortening, sugar, yeast nutrients, doughconditioners, and preservatives are blended to form a dough and aremixed to partial or full development. The resulting dough may be allowedto ferment for a period of time depending upon specific process ordesired end-product characteristics.

The next step in the process is the mechanical or manual division of thedough into appropriate size pieces of sufficient weight to ensureachieving the targeted net weight after baking, cooling, and slicing.The dough pieces are often then rounded and allowed to rest(Intermediate Proof) for varying lengths of time. This allows the doughto “relax” prior to sheeting and molding preparations. The timegenerally ranges from 5-15 minutes, but may vary considerably dependingon specific processing requirements and formulations. The dough piecesare then mechanically or manually formed into an appropriate shape arethen usually given a final “proof” prior to baking. The dough pieces arethen baked at various times, temperatures, and steam conditions in orderto achieve the desired end product.

In the Pre-Ferment method, yeast is combined with other ingredients andallowed to ferment for varying lengths of time prior to final mixing ofthe bread or roll dough. Baker's terms for these systems include “WaterBrew”, “Liquid Ferment”, “Liquid Sponge”, and “Sponge/Dough”. Apercentage of flour ranging from 0-100% is combined with the otheringredients which may include but are not limited to water, yeast, yeastnutrients and dough conditioners and allowed to ferment under controlledor ambient conditions for a period of time. Typical times range from 1-5hours. The ferment may then be used as is, or chilled and stored in bulktanks or troughs for later use. The remaining ingredients are added(flour, characterizing ingredients, additional additives, additionalwater, etc.) and the dough is mixed to partial or full development.

The dough is then allowed to ferment for varying time periods.Typically, as some fermentation has taken place prior to the addition ofthe remaining ingredients, the time required is minimal (i.e., 10-20min.), however, variations are seen depending upon equipment and producttype. Following the second fermentation step, the dough is then treatedas in the Straight Dough Method.

As used herein the term “dough mixture” describes a mixture thatminimally comprises a flour or meal and a liquid, such as milk or water.

As used herein the term “dough” describes an elastic, pliable proteinnetwork mixture that minimally comprises a flour, or meal and a liquid,such as milk or water.

As used herein the term “dough ingredient” may include, but is notexclusive of, any of the following ingredients: flour, water or otherliquid, grain, yeast, sponge, salt, shortening, sugar, yeast nutrients,dough conditioners and preservatives.

As used herein, the term “baked good” includes but is not exclusive ofall bread types, including yeast-leavened and chemically-leavened andwhite and variety breads and rolls, english muffins, cakes and cookies,confectionery coatings, crackers, doughnuts and other sweet pastrygoods, pie and pizza crusts, pretzels, pita and other flat breads,tortillas, pasta products, and refrigerated and frozen dough products.

While thioredoxin has been used to reduce albumins in flour, thiol redoxproteins have not been used to reduce glutenins and gliadins nor otherwater insoluble storage proteins, nor to improve the quality of doughand baked goods. Thiol redox proteins have also not been used to improvethe quality of gluten thereby enhancing its value nor to prepare doughfrom crop cereals such as barley, corn, sorghum, oat, millet and rice orfrom soybean flour.

Many cereal seeds also contain proteins that have been shown to act asinhibitors of enzymes from foreign sources. It has been suggested thatthese enzyme inhibitors may afford protection against certaindeleterious organisms (Garcia-Olmedo, F., et al. (1987), Oxford Surveysof Plant Molecular and Cell Biology 4:275-335; Birk, Y. (1976), Meth.Enzymol. 45:695-739, and Laskowski, M., Jr., et al. (1980), Ann. Reo.Biochem. 49:593-626). Two such type enzyme inhibitors are amylaseinhibitors and trypsin inhibitors. Furthermore, there is evidence that abarley protein inhibitor (not tested in this study) inhibits anα-amylase from the same source (Weselake, R. J., et al. (1983), PlantPhysiol. 72:809-812). Unfortunately, the inhibitor protein often causesundesirable effects in certain food products. The trypsin inhibitors insoybeans, notably the Kunitz trypsin inhibitor (KTI) and Bowman-Birktrypsin inhibitor (BBTI) proteins, must first be inactivated before anysoybean product can be ingested by humans or domestic animals. It isknown that these two inhibitor proteins become ineffective as trypsininhibitors when reduced chemically by sodium borohydride (Birk, Y.(1985), Int. J. Peptide Protein Res. 25:113-131, and Birk, Y. (1976),Meth. Enzymol. 45:695-739). These inhibitors like other proteins thatinhibit proteases contain intramolecular disulfides and are usuallystable to inactivation by heat and proteolysis (Birk (1976), supra.;Garcia-Olmedo, et al. (1987), supra., and Ryan (1980). Currently, tominimize the adverse effects caused by the inhibitors these soybeantrypsin inhibitors and other trypsin inhibitors in animal and human foodproducts are being treated by exposing the food to high temperatures.The heat treatment, however, does not fully eliminate inhibitoractivity. Further, this process is not only expensive but it alsodestroys many of the other proteins which have important nutritionalvalue. For example, while 30 min at 120° C. leads to completeinactivation of the BBTI of soy flour, about 20% of the original KTIactivity remains (Friedman, et al., 1991). The prolonged or highertemperature treatments required for full inactivation of inhibitorsresults in destruction of amino acids such as cystine, arginine, andlysine (Chae, et al., 1984; Skrede and Krogdahl, 1985).

There are also several industrial processes which require α-amylaseactivity. One example is the malting of barley which requires activeα-amylase. Inactivation of inhibitors such as the barleyamylase/subtilisin (asi) inhibitor and its equivalent in other cerealsby thiol redox protein reduction would enable α-amylases to become fullyactive sooner than with present procedures, thereby shortening time formalting or similar processes.

Thiol redox proteins have also not previously been used to inactivatetrypsin or amylase inhibitor proteins. The reduction of trypsininhibitors such as the Kunitz and Bowman-Birk inhibitor proteinsdecreases their inhibitory effects (Birk, Y. (1985), Int. J. PeptideProtein Res. 25:113-131). A thiol redox protein linked reduction of theinhibitors in soybean products designed for consumption by humans anddomestic animals would require no heat or lower heat than is presentlyrequired for protein denaturization, thereby cutting the costs ofdenaturation and improving the quality of the soy protein. Also aphysiological reductant, a so-called clean additive (i.e., an additivefree from ingredients viewed as “harmful chemicals”) is highly desirablesince the food industry is searching for alternatives to chemicaladditives. Further the ability to selectively reduce the major wheat andseed storage proteins which are important for flour quality (e.g., thegliadins and the glutenins) in a controlled manner by a physiologicalreductant such as a thiol redox protein would be useful in the bakingindustry for improving the characteristics of the doughs from wheat andrye and for creating doughs from other grain flours such as cerealflours or from cassava or soybean flour.

The family of 2S albumin proteins characteristic of oil-storing seedssuch as castor bean and Brazil nut (Kreis, et al. 1989; Youle and Huang,1981) which are housed within protein bodines in the seed endosperm orcotyledons (Ashton, et al. 1976; Weber, et al. 1980), typically consistof dissimilar subunits connected by two intermolecular disculfidebonds—one subunit of 7 to 9 kDa and the other of 3 to 4 kDa. The largesubunit contains two intramolecular disulfide groups, the small subunitcontains none. The intramolecular disulfides of the 2S large subunitshow homology with those of the soybean Bowman-Birk inhibitor (Kreis, etal. 1989) but nothing is known of the ability of 2S proteins to undergoreduction under physiological conditions.

These 2S albumin proteins are rich in methionine. Recently transgenicsoybeans which produce Brazil nut 2S protein have been generated.Reduction of the 2S protein in such soybeans could enhance theintegration of the soy proteins into a dough network resulting in asoybread rich in methionine. In addition, these 2S proteins are oftenallergens. Reduction of the 2S protein would result in the cessation ofits allergic activity.

Pullulanase (“debranching enzyme”) is an enzyme that breaks down thestarch of the endosperm of cereal seeds by hydrolytically cleaving α-1,6bonds. Pullulanase is an enzyme fundamental to the brewing and bakingindustries. Pullulanase is required to break down starch in malting andin certain baking procedures carried out in the absence of added sugarsor other carbohydrates. obtaining adequate pullulanase activity is aproblem especially in the malting industry. It has been known for sometime that dithiothreitol (DTT, a chemical reductant for thioredoxin)activates pullulanase of cereal preparations (e.g., barley, oat and riceflours). A method for adequately activating or increasing the activityof pullulanase with a physiologically acceptable system, could lead tomore rapid malting methods and, owing to increased sugar availability,to alcoholic beverages such as beers with enhanced alcoholic content.

Death and permanent injury resulting from snake bites are seriousproblems in many African, Asian and South American countries and also amajor concern in several southern and western areas of the UnitedStates. Venoms from snakes are characterized by active proteincomponents (generally several) that contain disulfide (S-S) bridgeslocated in intramolecular (intrachain) cystines and in some cases inintermolecular (interchain) cystines. The position of the cystine withina given toxin group is highly conserved. The importance ofintramolecular S-S groups to toxicity is evident from reports showingthat reduction of these groups leads to a loss of toxicity in mice(Yang, C. C. (1967) Biochim. Biophys. Acta. 133:346-355; Howard, B. D.,et al. (1977) Biochemistry 16:122-125). The neurotoxins of snake venomare proteins that alter the release of neurotransmitter from motor nerveterminals and can be presynaptic or postsynaptic. Common symptomsobserved in individuals suffering from snake venom neurotoxicity includeswelling, edema and pain, fainting or dizziness, tingling or numbing ofaffected part, convulsions, muscle contractions, renal failure, inaddition to long-term necrosis and general weakening of the individual,etc.

The presynaptic neurotoxins are classified into two groups. The firstgroup, the β-neurotoxins, include three different classes of proteins,each having a phospholipase A₂ component that shows a high degree ofconservation. The proteins responsible for the phospholipase A₂ activityhave from 6 to 7 disulfide bridges. Members of the β-neurotoxin groupare either single chain (e.g., caudotoxin, notexin and agkistrodotoxin)or multichain (e.g., crotoxin, ceruleotoxin and Vipera toxin).β-bungarotoxin, which is made up of two subunits, constitutes a thirdgroup. One of these subunits is homologous to the Kunitz-type proteinaseinhibitor from mammalian pancreas. The multichain β-neurotoxins havetheir protein components linked ionically whereas the two subunits ofβ-bungarotoxin are linked covalently by an intermolecular disulfide. TheB chain subunit of β-bungarotoxin, which is also homologous to theKunitz-type proteinase inhibitor from mammalian pancreas, has 3disulfide bonds.

The second presynaptic toxin group, the facilitatory neurotoxins, isdevoid of enzymatic activity and has two subgroups. The first subgroup,the dendrotoxins, has a single polypeptide sequence of 57 to 60 aminoacids that is homologous with Kunitz-type trypsin inhibitors frommammalian pancreas and blocks voltage sensitive potassium channels. Thesecond subgroup, such as the fasciculins (e.g., fasciculin 1 andfasiculin 2) are cholinesterase inhibitors and have not been otherwiseextensively studied.

The postsynaptic neurotoxins are classified either as long or shortneurotoxins. Each type contains S-S groups, but the peptide is uniqueand does not resemble either phospholipase A₂ or the Kunitz orKunitz-type inhibitor protein. The short neurotoxins (e.g., erabutoxin aand erabutoxin b) are 60 to 62 amino acid residues long with 4intramolecular disulfide bonds. The long neurotoxins (e.g.,α-bungarotoxin and α-cobratoxin) contain from 65 to 74 residues and 5intramolecular disulfide bonds. Another type of toxins, the cytotoxins,acts postsynaptically but its mode of toxicity is ill defined. Thesecytotoxins show obscure pharmacological effects, e.g., hemolysis,cytolysis and muscle depolarization. They are less toxic than theneurotoxins. The cytotoxins usually contain 60 amino acids and have 4intramolecular disulfide bonds. The snake venom neurotoxins all havemultiple intramolecular disulfide bonds.

The current snake antitoxins used to treat poisonous snake bitesfollowing first aid treatment in individuals primarily involveintravenous injection of antivenom prepared in horses. Although it isnot known how long after envenomation the antivenom can be administeredand be effective, its use is recommended up to 24 hours. Antivenomtreatment is generally accompanied by administration of intravenousfluids such as plasma, albumin, platelets or specific clotting factors.In addition, supporting medicines are often given, for example,antibiotics, antihistamines, antitetanus agents, analgesics andsedatives. In some cases, general treatment measures are taken tominimize shock, renal failure and respiratory failure. Other thanadministering calcium-EDTA in the vicinity of the bite and excising thewound area, there are no known means of localized treatment that resultin toxin neutralization and prevention of toxic uptake into the bloodstream. Even these localized treatments are of questionable significanceand are usually reserved for individuals sensitive to horse serum(Russell, F. E. (1983) Snake Venom Poisoning, Schollum International,Inc. Great Neck, N.Y.).

The term “individual” as defined herein refers to an animal or a human.

Most of the antivenoms in current use are problematic in that they canproduce harmful side effects in addition to allergic reactions inpatients sensitive to horse serum (up to 5% of the patients).Nonallergic reactions include pyrogenic shock, and complement depletion(Chippaur, J.-P., et al. (1991) Reptile Venoms and Toxins, A. T. Tu,ed., Marcel Dekker, Inc., pp. 529-555).

It has been shown that thioredoxin, in the presence of NADPH andthioredoxin reductase reduces the bacterial neurotoxins tetanus andbotulinum A in vitro (Schiavo, G., et al. (1990) Infection and Immunity58:4136-4141; Kistner, A., et al. (1992) Naunyn-Schmiedeberg's ArchPharmacol 345:227-234). Thioredoxin was effective in reducing theinterchain disulfide link of tetanus toxin and such reduced tetanustoxin was no longer neurotoxic (Schiavo, et al., supra.). However,reduction of the interchain disulfide of botulinum A toxin bythioredoxin was reported to be much more sluggish (Kistner, et al.,supra.). In contrast to the snake neurotoxin studied in the course ofthis invention, the tetanus research group (Schiavo, et al., supra.)found no evidence in the work done with the tetanus toxin that reducedthioredoxin reduced toxin intrachain disulfide bonds. There was also noevidence that thioredoxin reduced intrachain disulfides in the work donewith botulinum A. The tetanus and botulinum A toxins are significantlydifferent proteins from the snake neurotoxins in that the latter (1)have a low molecular weight; (2) are rich in intramolecular disulfidebonds; (3) are resistant to trypsin and other animal proteases; (4) areactive without enzymatic modification, e.g., proteolytic cleavage; (5)in many cases show homology to animal proteins, such as phospholipase A₂and Kunitz-type proteases; (6) in most cases lack intermoleculardisulfide bonds, and (7) are stable to agents such as heat andproteases.

Reductive inactivation of snake toxins in vitro by incubation with 1%β-mercaptoethanol for 6 hours and incubation with 8M urea plus 300 mMβ-mercaptoethanol has been reported in the literature (Howard, B. D., etal. (1977) Biochemistry 16:122-125; Yang, C. C. (1967) Biochim. Biophys.Acta. 133:346-355). These conditions, however, are far fromphysiological. As defined herein the term “inactivation” with respect toa toxin protein means that the toxin is no longer biologically active invitro, in that the toxin is unable to link to a receptor. Also as usedherein, “detoxification” is an extension of the term “inactivation” andmeans that the toxin has been neutralized in an individual as determinedby animal toxicity tests.

Bee venom is a complex mixture with at least 40 individual components,that include major components as melittin and phospholipase A₂,representing respectively 50% and 12% of the total weight of the venom,and minor components such as small proteins and peptides, enzymes,amines, and amino acids.

Melittin is a polypeptide consisting of 26 amino acids with a molecularweight of 2840. It does not contain a disulfide bridge. Owing to itshigh affinity for the lipid-water interphase, the protein permeates thephospholipid bilayer of the cell membranes, disturbing its organizedstructure. Melittin is not by itself a toxin but it alters the structureof membranes and thereby increases the hydrolytic activity ofphospholipase A₂, the other major component and the major allergenpresent in the venom.

Bee venom phospholipase A₂ is a single polypeptide chain of 128 aminoacids, is cross-linked by four disulfide bridges, and containscarbohydrate. The main toxic effect of the bee venom is due to thestrong hydrolytic activity of phospholipase A₂ achieved in associationwith melittin.

The other toxic proteins in bee venom have a low molecular weight andcontain at least two disulfide bridges that seem to play an importantstructural role. Included are a protease inhibitor (63-65 amino acids),MCD or 401-peptide (22 amino acids) and apamin (18 amino acids).

Although there are thousands of species of bees, only the honey bee,Apis mellifera, is a significant cause of allergic reactions. Theresponse ranges from local discomfort to systemic reactions such asshock, hypotension, dyspnea, loss of consciousness, wheezing and/orchest tightness that can result in death. The only treatment that isused in these cases is the injection of epinephrine.

The treatment of bee stings is important not only for individuals withallergic reactions. The “killer” or Africanized bee, a variety of honeybee is much more aggressive than European honey bees and represents adanger in both South and North America. While the lethality of the venomfrom the Africanized and European bees appears to be the same(Schumacher, M. I., et al. (1989) Nature 337:413), the behaviour patternof the hive is completely different. It was reported that Africanizedbees respond to colony disturbance more quickly, in greater numbers andwith more stinging (Collins, A. M., et al. (1982) Science 218:72-74). Amass attack by Africanized bees may produce thousands of stings on oneindividual and cause death. The “killer” bees appeared as a result ofthe interbreeding between the African bee (Apis mellifera scutellata)and the European bee (Apis mellifera mellifera). African bees wereintroduced in 1956 into Brazil with the aim of improving honeyproduction being a more tropically adapted bee. Africanized bees havemoved from South America to North America, and they have been reportedin Texas and Florida.

In some areas of the world such as Mexico, Brazil, North Africa and theMiddle East, scorpions present a life hazard to humans. However, onlythe scorpions of family Buthidae (genera, Androctonus, Buthus,Centruroides, Leiurus and Tityus) are toxic for humans. The chemicalcomposition of the scorpion venom is not as complex as snake or beevenom. Scorpion venom contains mucopolysaccharides, small amounts ofhyaluronidase and phospholipase, low molecular-weight molecules,protease inhibitors, histamine releasers and neurotoxins, such asserotonin. The neurotoxins affect voltage-sensitive ionic channels inthe neuromuscular junction. The neurotoxins are basic polypeptides withthree to four disulfide bridges and can be classified in two groups:peptides with from 61 to 70 amino acids, that block sodium channel, andpeptides with from 36 to 39 amino acids, that block potassium channel.The reduction of disulfide bridges on the neurotoxins bynonphysiological reductants such as DTT or β-mercaptoethanol (Watt, D.D., et al. (1972) Toxicon 10:173-181) lead to loss of their toxicity.

Symptoms of animals stung by poisonous scorpions includehyperexcitability, dyspnea, convulsions, paralysis ad death. At present,antivenin is the only antidote for scorpion stings. The availability ofthe venom is a major problem in the production of antivenin. Unlikesnake venom, scorpion venom is very difficult to collect, because theyield of venom per specimen is limited and in some cases the storage ofdried venom leads to modification of its toxicity. An additional problemin the production of antivenins is that the neurotoxins are very poorantigens.

The reductive inactivation of snake, bee and scorpion toxins underphysiological conditions has never been reported nor has it beensuggested that the thiol redox agents, such as reduced lipoic acid, DTT,or reduced thioredoxin could act as an antidote to these venoms in anindividual.

SUMMARY OF THE INVENTION

It is an object herein to provide a method for reducing a non thionincystine containing protein.

It is a second object herein to provide methods utilizing a thiol redoxprotein alone or in combination with a reductant or reduction system toreduce glutenins or gliadins present in flour or seeds.

It is also an object herein to provide methods using a thiol redoxprotein alone or in combination with a reductant or reduction system toimprove dough strength and baked goods characteristics such as bettercrumb quality, softness of the baked good and higher loaf volume.

It is a further object herein to provide formulations containing a thiolredox protein useful in practicing such methods.

Still a further object herein is to provide a method for producing adough from rice, corn, soybean, barley, oat, cassava, sorghum or milletflour.

Yet, another object is to provide a method for producing an improvedgluten or for producing a gluten-like product from cereal grains otherthan wheat and rye.

It is further an object herein to provide a method of reducing an enzymeinhibitor protein having disulfide bonds.

Still another object herein is to provide yeast cells geneticallyengineered to express or overexpress thioredoxin.

Still yet another object herein is to provide yeast cells geneticallyengineered to express or overexpress NADP-thioredoxin reductase.

Still yet a further object herein is to provide a method for improvingthe quality of dough or a baked good using such genetically engineeredyeast cells.

Yet still another object herein is to provide a method of reducing theintramolecular disulfide bonds of a non-thionin, non chloroplast proteincontaining more than one intramolecular cystine comprising adding athiol redox protein to a liquid or substance containing the cystinescontaining protein, reducing the thiol redox protein and reducing thecystines containing protein by means of the thiol redox protein.

Another object herein is to provide an isolated pullulanase inhibitorprotein having disulfide bonds and a molecular weight of between 8 to 15kDa.

Still another object herein is to provide a method of increasing theactivity of pullulanase derived from barley or wheat endospermcomprising adding thioredoxin to a liquid or substance containing thepullulanase and reducing the thioredoxin thereby increasing thepullulanase activity.

Still another object herein is to provide a method of reducing an animalvenom toxic protein having one or more intramolecular cystinescomprising contacting the cystine containing protein with an amount of athiol redox (SH) agent effective for reducing the protein, andmaintaining the contact for a time sufficient to reduce one or moredisulfide bridges of the one or more intramolecular cystines therebyreducing the neurotoxin protein. The thiol redox (SH) agent may be areduced thioredoxin, reduced lipoic acid in the presence of athioredoxin, DTT or DTT in the presence of a thioredoxin and the snakeneurotoxin protein may be a presynaptic or postsynaptic neurotoxin.

Still a further object of the invention is to provide a compositioncomprising a snake neurotoxin protein and a thiol redox (SH) agent.

Still yet another object of the invention is to provide a method ofreducing an animal venom toxic protein having one or more intramolecularcystines comprising contacting the protein with amounts ofNADP-thioredoxin reductase, NADPH or an NADPH generator system and athioredoxin effective for reducing the protein, and maintaining thecontact for a time sufficient to reduce one or more disulfide bridges ofthe one or more intramolecular cystines thereby reducing the protein.

Yet another object herein is to provide a method of inactivating, invitro, a snake neurotoxin having one or more intramolecular cystinescomprising adding a thiol redox (SH) agent to a liquid containing thetoxin wherein the amount of the agent is effective for reducing thetoxin.

Yet a further object herein is to provide a method of treating venomtoxicity in an individual comprising administering, to an individualsuffering from venom toxicity, amounts of a thiol redox (SH) agenteffective for reducing or alleviating the venom toxicity.

In accordance with the objects of the invention, methods are providedfor improving dough characteristics comprising the steps of mixing athiol redox protein with dough ingredients to form a dough and bakingsaid dough.

Also, in accordance with the objects of the invention, a method isprovided for inactivating an enzyme inhibitor protein in a grain foodproduct comprising the steps of mixing a thiol redox protein with theseed product, reducing the thiol redox protein by a reductant orreduction system and reducing the enzyme inhibitor by the reduced thiolredox protein, the reduction of the enzyme inhibitor inactivating theenzyme inhibitor.

The thiol redox proteins in use herein can include thioredoxin andglutaredoxin. The thioredoxin includes but is not exclusive of E. colithioredoxin, thioredoxin h, f and m and animal thioredoxins. A reductantof thioredoxin used herein can include lipoic acid or a reduction systemsuch as NADPH in combination with NADP thioredoxin reductase (NTR). Thereductant of glutaredoxin can include reduced glutathione in conjunctionwith the reduction system NADPH and glutathione reductase. NADPH can bereplaced with an NADPH generator or generator composition such as oneconsisting of glucose 6-phosphate, NADP and glucose 6-phosphatedehydrogenase from a source such as yeast. The NADPH generator is addedtogether with thioredoxin and NADP-thioredoxin reductase at the start ofthe dough making process.

It should be noted that the invention can also be practiced withcysteine containing proteins. The cysteines can first be oxidized andthen reduced via thiol redox protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a graph showing the effect of α-amylase proteininhibitors on activation of NADP-Malate Dehydrogenase in the presence ofDTT-reduced Thioredoxin h.

FIG. 2 is a graph showing the effect of α-amylase InhibitorConcentration on NADP-Malate Dehydrogenase Activation by α-amylaseInhibitors.

FIG. 3 is a graph showing the effect of Thioredoxin h Concentration onActivation of NADP-Malate dehydrogenase by DSG-1 or -2 α-AmylaseInhibitors.

FIG. 4 is a graph showing the effect of α-Amylase Inhibitors on DTNBReduction by the E. coli NADP/Thioredoxin System.

FIG. 5 is a graph showing the effect of purothionin a and CM-1 α-AmylaseInhibitor from Bread Wheat on DTNB Reduction by the E. coliNADP/Thioredoxin System.

FIG. 6 is a photograph taken of an SDS polyacrylamide electrophoreticgel placed over a long UV wavelength light box showing theThioredoxin-Linked Reduction of Soluble Sulfur Rich Seed Proteins: DurumWheat α-Amylase Inhibitor (DSG-1) and Bowman-Birk Soybean TrypsinInhibitor (BBTI).

FIG. 7 is a photograph taken of an SDS polyacrylamide electrophoreticgel placed over a long UV wavelength light box showing theThioredoxin-Linked Reduction of Seed Proteins.

FIG. 8 is a photograph taken of an SDS-polyacrylamide electrophoreticgel placed over a fluorescent light box showing Thioredoxin-linkedreduction of gliadins determined by an SDS-PAGE/mBBr procedure.

FIG. 9 is a photograph taken of an acidic-polyacrylamide electrophoreticgel placed over a fluorescent light box showing Thioredoxin-linkedreduction of the different types of gliadins determined by an acidPAGE/mBBr procedure.

FIG. 10 is a photograph taken of an SDS-polyacrylamide electrophoreticgel placed over a fluorescent light box showing Thioredoxin-linkedreduction of acid soluble glutenins determined by an SDS-PAGE/mBBrprocedure.

FIG. 11 is a graph showing the relative reduction of seed proteinfractions during germination.

FIG. 12 is a bar graph showing reduction of principal thioredoxin-linkedgliadins and glutenins during germination (compared with in vivoreduction).

FIG. 13 is a diagrammatic representation of the proposed role ofthioredoxin in forming a protein network for bread and pasta.

FIG. 14 shows farinograms of treated and untreated medium quality flour;FIG. 14(a) is the farinogram of the medium quality flour; FIG. 14(b) isof the same flour following treatment with reduced glutathione, and FIG.14(c) is of the medium quality flour after treatment with theNADP/thioredoxin system.

FIG. 15 shows farinograms of treated and untreated poor quality flour;FIG. 15(a) is a farinogram of the poor quality flour control; FIG. 15(b)is of the same flour after treatment with reduced glutathione, and FIG.15(c) is of the poor quality flour after treatment with DTT, and FIG.15(d) is of the poor quality flour after treatment with theNADP/thioredoxin system.

FIG. 16 shows farinograms of treated and untreated Apollo flour; FIG.16(a) represents the untreated flour, and FIG. 16(b) represents the sameflour treated with the NTS.

FIG. 17 shows farinograms of treated and untreated Apollo flour; FIG.17(a) is a farinogram of the Apollo control; FIG. 17(b) is of the sameflour after treatment with an NADPH generating system; FIG. 17(c) is ofthe Apollo flour after treatment with the same generating system plusNTR and thioredoxin.

FIG. 18 is a photograph showing a top view of a comparison between anArbon loaf of bread made from thioredoxin-treated dough and an untreatedcontrol loaf.

FIG. 19 is a photograph showing a side elevational and partial top viewof a comparison between a loaf made from a thioredoxin-treated Arbonflour dough and an untreated control loaf.

FIG. 20 is a photograph showing a side elevational view of a comparisonbetween a loaf made from a thioredoxin-treated Arbon flour dough and anuntreated control loaf.

FIG. 21 is a photograph showing a top view of a comparison between aloaf made from a thioredoxin-treated Arbon flour dough and an untreatedcontrol loaf.

FIG. 22 is a photograph showing a side elevational and partial top viewof a comparison between a loaf made from a thioredoxin-treated Arbonflour dough and an untreated control loaf.

FIG. 23 shows photographs comparing breads baked from thioredoxintreated and untreated doughs; FIG. 23(a) shows a comparison of loaves ofbread made from treated and untreated arbon flour, and FIG. 23(b) showsa comparison among baked goods that were prepared fromthioredoxin-treated and untreated corn, rice and sorghum flour.

FIG. 24 is a photograph showing a top and partial side view of acomparison between a loaf baked from a triticale flour dough treatedwith thioredoxin and a control loaf made from untreated triticale flourdough.

FIG. 25 is a photograph showing comparisons among baked goods that wereprepared from thioredoxin-treated and untreated corn, rice and sorghumflour.

FIG. 26 represents photographs of an SDS polyacrylamide electrophoreticgel showing the extent of thioredoxin-linked reduction ofmyristate-extracted proteins from oat flour.

FIG. 27 represents photographs of an SDS polyacrylamide electrophoreticgel showing the extent of thioredoxin-linked reduction ofmyristate-extracted proteins from triticale flour.

FIG. 28 represents photographs of an SDS polyacrylamide electrophoreticgel showing the extent of thioredoxin-linked reduction ofmyristate-extracted proteins from rye flour.

FIG. 29 represents photographs of an SDS polyacrylamide electrophoreticgel showing the extent of thioredoxin-linked reduction ofmyristate-extracted proteins from barley flour.

FIG. 30 represents photographs of an SDS polyacrylamide electrophoreticgel showing the extent of thioredoxin-linked reduction of buffer,ethanol and myristate-extracted proteins from teff flour; FIG. 30(a)shows fluorescence and FIG. 30(b) shows the protein staining.

FIG. 31 is a photograph of an SDS polyacrylamide electrophoretic gelshowing the effect of NTS vs. glutathione reductase on the reductionstatus of myristate-extracted proteins from corn, sorghum and rice.

FIG. 32 is a photograph of an SDS polyacrylamide electrophoretic gelshowing the in vivo reduction status and thioredoxin-linked in vitroreduction of myristate-extracted proteins from corn, sorghum and rice.

FIG. 33 represents photographs of an SDS polyacrylamide electrophoreticgel showing the relative reduction of wheat glutenins by a yeastNADP/thioredoxin system.

FIG. 34 represents photographs of an SDS polyacrylamide electrophoreticgel showing the relative reduction of wheat gliadins by a yeastNADP/thioredoxin system.

FIG. 35 represents photographs of an SDS polyacrylamide electrophoreticgel showing the extent of thioredoxin-linked reduction ofethanol-extracted proteins from triticale flour.

FIG. 36 represents photographs of an SDS polyacrylamide electrophoreticgel showing the extent of thioredoxin-linked reduction ofethanol-extracted proteins from rye flour.

FIG. 37 represents photographs of an SDS polyacrylamide electrophoreticgel showing the extent of thioredoxin-linked reduction ofethanol-extracted proteins from oat flour.

FIG. 38 represents photographs of an SDS polyacrylamide electrophoreticgel showing the extent of thioredoxin-linked reduction ofethanol-extracted proteins from barley flour.

FIG. 39 represents photographs of an SDS polyacrylamide electrophoreticgels showing the extent of reduction of castor seed matrix andcrystalloid proteins by various reductants.

FIG. 40 is a photograph of an SDS polyacrylamide electrophoretic gelshowing the reduction specificity of 2S proteins.

FIG. 41 is a graph showing the separation of pullulanase inhibitor frompullulanase of barley malt by DE52 chromatography.

FIG. 42 is a graph showing the purification of pullulanase inhibitor ofbarley malt by CM32 chromatography at pH 4.6.

FIG. 43 is a graph showing the purification of pullulanase inhibitor ofbarley malt by CM32 chromatography at pH 4.0.

FIG. 44 is a graph showing the purification of pullulanase inhibitor ofbarley malt by Sephadex G-75 chromatography.

FIG. 45 represents photographs of SDS polyacrylamide electrophoreticgels showing the extent of reduction of bee venom proteins by variousreductants.

FIG. 46 represents photographs of SDS polyacrylamide electrophoreticgels showing the extent of reduction of scorpion venom proteins byvarious reductants.

FIG. 47 represents photographs of SDS polyacrylamide electrophoreticgels showing the extent of reduction of snake venom proteins by variousreductants.

FIG. 48 represents photographs of an SDS polyacrylamide electrophoreticgel showing the extent of reduction of bee, scorpion and snake venomproteins with the NTS in the presence and absence of proteaseinhibitors.

FIG. 49 is a photograph of an SDS polyacrylamide electrophoretic gelshowing the extent of reduction of erabutoxin b samples treated withdifferent reductants.

FIG. 50 is a graph showing the activation of chloroplast NADP-malatedehydrogenase by erabutoxin b reduced with different thioredoxinscompared to the activation of the dehydrogenase by a control lackingtoxin.

FIG. 51 is a graph showing the effect of thioredoxin-linked reduction ofβ-bungarotoxin on β-bungarotoxin phospholipase A₂ activity.

FIG. 52 is a photograph of an SDS polyacrylamide electrophoretic gelshowing the extent of reduction of bungarotoxin and α-bungarotoxinsamples with cellular reductants.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with this detailed description, the following definitionsand abbreviations apply:

CM - certain bread wheat α-amylase inhibitors DSG - certain α-amylaseinhibitors isolated from  durum wheat DTNB - 2′5′-dithiobis(2-nitrobenzoic acid) NTR - NADP-thioredoxin reductase mBBr -monobromobimane NADP-MDH - NADP-malate dehydrogenase FBPase -fructose-1,6-bisphosphatase SDS - sodium dodecyl sulfate DTT -dithiothreitol Cereal - millet, wheat, oat, barley, rice, sorghum,  orrye BBTI - Bowman-Birk soybean trypsin inhibitor KTI - Kunitz soybeantrypsin inhibitor PAGE - polyacrylamide gel electrophoresis TCA -trichloroacetic acid

Enzyme Inhibitor Protein Experiments Starting Materials

Materials

Seeds of bread wheat Triticum aestivum L, cv. Talent) and durum wheat(Triticum durum. Desf., cv. Mondur) were obtained from laboratorystocks.

Reagents

Chemicals and fine chemicals for enzyme assays and sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis were purchased fromSigma Chemical Co. and BioRad Laboratories, respectively.Monobromobimane (mBBr, tradename Thiolite) was purchased fromCalbiochem. Other chemicals were obtained from commercial sources andwere of the highest quality available.

Enzymes

Thioredoxin and NTR from E. coli ware purchased from AmericanDiagnostics, Inc. and were also isolated from cells transformed tooverexpress each protein. The thioredoxin strain containing therecombinant plasmid, pFP1, was kindly provided by Dr. J.-P. Jacquot (dela Motte-Guery, F. et al. (1991) Eur. J. Biochem. 196:287-294). The NTRstrain containing the recombinant plasmid, pPMR21, was kindly providedby Drs. Marjorie Russel and Peter Model (Russel, M. et al. (1988) J.Biol. Chem. 263:9015-9019). The Isolation procedure used for theseproteins was as described in those studies with the following changes:cells were broken in a Ribi cell fractionator at 25,000 psi and NTR waspurified as described by Florencio et al. (Fiorencio, F. J. et al.(1988) Arch. Biochem. Biophys. 266:496-507) without the red agarose.Thioredoxin and NTR from Saccharomyces cerevisiae (baker's yeast type 1)were isolated by the procedure developed by Florencio, et al. forspinach leaves with the following changes: suspended cells [1 partcells:5 parts buffer (w/v)], were broken in a Ribi cell fractionator at40,000 psi with three passes.

Thioredoxin h and NTR were isolated from wheat germ by the proceduredeveloped for spinach leaves (Florencio, F. J., et al. (1988), Arch.Biochem. Biophys. 26:496-507). NADP-malate dehydrogenase (NADP-MDH) andfructose-1,6-bisphosphatase (FBPase) were purified from leaves of corn(Jacquot, J.-P., et al. (1981), Plant Physiol. 68:300-304) and spinach(Crawford, N. A., et al. (1989), Arch. Biochem. Biophys. 217:223-239)respectively. E. coli glutaredoxin and calf thymus thioredoxin wereobtained from Professor A. Holmgren.

α-Amylase and Trypsin Inhibitors

CM-1 protein was isolated from the albumin-globulin fraction of breadwheat flour as described previously. (Kobrehel, K., et al. (1991),Cereal Chem. 68:1-6). A published procedure was also used for theisolation of DSG proteins (DSG-1 and DSG-2) from the glutenin fractionof durum wheat (Kobrehel, K. et al. (1989), J. Sci. Food Agric.48:441-452). The CM-1, DSG-1 and DSG-2 proteins were homogeneous inSDS-polyacrylamide gel electrophoresis. Trypsin inhibitors werepurchased from Sigma Chemical Co., except for the one from corn kernelwhich was from Fluka. In all cases, the commercial preparations showed asingle protein component which migrated as expected in SDS-PAGE(Coomassie Blue stain), but in certain preparations, the band was notsharp.

Other Proteins

Purothionin a from bread wheat and purothionins α-1 and β from durumwheat were kind gifts from Drs. D. D. Kasarda and B. L. Jones,respectively. The purothionin α sample contained two members of thepurothionin family when examined with SDS-polyacrylamide gelelectrophoresis. The purothionin α-1 and β samples were both homogeneousin SDS-polyacrylamide gel electrophoresis.

Routine Method Steps

Enzyme Activation Assays

The NADP-MDH, FBPase, NTR and Thioredoxin h assay methods were accordingto Florencio, F. J., et al. (1988), Arch. Biochem. Biophys. 266:496-507with slight modifications as indicated. For enzyme activation assays,the preincubation time was 20 min. unless specified otherwise.

mBBr Fluorescent Labeling and SDS-polyacrylamide Gel ElectrophoresisAnalyses

Direct reduction of the proteins under study was determined by amodification of the method of Crawford, et al. (Crawford, N. A., et al.(1989), Arch. Biochem. Biophys. 271:223-239). The reaction was carriedout in 100 mM potassium phosphate buffer, pH 7.1, containing 10 mM EDTAand 16% glycerol in a final volume of 0.1 ml. As indicated, 0.7 μg (0.1AM) NTR and 1 μg (0.8 μM) thioredoxin (both routinely from E. coli wereadded to 70 μl of the buffer solution containing 1 mM NADPH and 10 μg (2to 17 μM) of target protein. When thioredoxin was reduced bydithiothreitol (DTT, 0.5 mM), NADPH and NTR were omitted. Assays withreduced glutathione were performed similarly, but at a finalconcentration of 1 mM. After incubation for 20 min, 100 nmoles of mBBrwere added and the reaction was continued for another 15 min. To stopthe reaction and derivatize excess mBBr, 10 μl of 10% SDS and 10 μl of100 mM β-mercaptoethanol were added and the samples were then applied tothe gels. In the case of reduction by glutaredoxin, the thioredoxin andNTR were replaced by 1 μg (0.8 μM) E. coli glutaredoxin, 1.4 μg (0.14μM) glutathione reductase purified from spinach leaves (Florencio, F.J., et al. (1988), Arch. Biochem. Biophys. 266:496-507) and 1.5 mM NADPHwas used.

Gels (17.5% w/v, 1.5 mm thickness) were prepared according to Laemmli(Laemmli, U. K. (1970), Nature 227:680-685) and developed for 16 hr. atconstant current (9 mA). Following electrophoresis, gels were placed ina solution of 40% methanol and 10% acetic acid, and soaked for 4 to 6hours with several changes of the solution. Gels were then examined forfluorescent bands with near ultraviolet light and photographed (exposuretime 25 sec) according to Crawford, et al. (Crawford, N. A., et al.(1989), Arch. Biochem. Biophys. 271:223-239). Finally, gels were stainedwith Coomassie Blue and destained as before (Crawford, N. A., et al.(1989), Arch. Biochem. Biophys. 271:223-239).

Quantification of Labeled Proteins

To obtain a quantitative indication of the extent of reduction of testproteins by the NADP/thioredoxin system, the intensities of theirfluorescent bands seen in SDS-polyacrylamide gel electrophoresis wereevaluated, using a modification of the procedure of Crawford, et al.(Crawford, N. A., et al. (1989), Arch. Biochem. Biophys. 271:223-239).The photographic negatives were scanned using a Pharmacia Ultrascanlaser densitometer, and the area underneath the peaks was quantitated bycomparison to a standard curve determined for each protein. For thelatter determination, each protein (at concentrations ranging from 1 to5 μg) was reduced by heating for 3 min. at 100° C. in the presence of0.5 mM DTT. Labeling with mBBr was then carried out as described aboveexcept that the standards were heated for 2 min. at 100° C. after thereaction was stopped with SDS and excess mBBr derivatized withβ-mercaptoethanol.

Because the intensity of the fluorescent bands was proportional to theamounts of added protein, it was assumed that reduction was completeunder the conditions used.

EXAMPLE 1 Thioredoxin-linked Reduction of α-Amylase Inhibitors

Enzyme Activation Assays

The capability to replace a specific thioredoxin in the activation ofchloroplast enzymes is one test for the ability of thiol groups of agiven protein to undergo reversible redox change. Even though notphysiological in the case of extraplastidic proteins, this test hasproved useful in several studies. A case in point is purothionin which,when reduced by thioredoxin h activates chloroplast FBPase (Wada, K. etal. (1981), FEBS Lett., 124:237-240, and Johnson, T. C., et al. (1987),Plant Physiol., 85:446-451). The FBPase, whose physiological activatoris thioredoxin f, is unaffected by thioredoxin h. In this Example, theability of cystine-rich proteins to activate FBPase as well as NADP-MDHwas tested as set forth above. The α-amylase inhibitors from durum wheat(DSG-1 and DSG-2) were found to be effective in enzyme activation;however, they differed from purothionin in showing a specificity forNADP-MDH rather than FBPase (Table I). The α-amylase inhibitors wereactive only in the presence of reduced thioredoxin h, which itself didnot significantly activate NADP-MDH under these conditions (FIG. 1). Asshown in FIG. 1, DSG-1 and DSG-2 activated NADP-malate dehydrogenase inthe presence of DTT-reduced thioredoxin h according to the reactionsequence (DTT→Thioredoxin→DSG→NADP-MDH).

FIG. 1 represents results obtained with either the DSG-1 or DSG-2inhibitors from durum wheat; β-MET refers to β-mercaptoethanol. Thecomplete system for activation contained in 200 μl of 100 mM Tris-HClbuffer, pH 7.9; 10 mM DTT; 0.7 μg corn leaf NADP-MDH; 0.25 μg wheatthioredoxin h and 10 μg of DSG-1 or DSG-2. As indicated, 20 mMβ-mercaptoethanol (β-MET) replaced DTT. Following preincubation,NADP-MDH was assayed spectrophoto-metrically.

In the enzyme activation assays, thioredoxin h was reduced by DTT; asexpected, monothiols such as β-mercaptoethanol (β-MET), which do notreduce thioredoxin at a significant rate under these conditions(Jacquot, J.-P., et al. (1981), Plant Physiol. 68:300-304; Nishizawa, A.N., et al. (1982), “Methods in Chloroplast Molecular Biology”, (M.Edelman, R. B. Hallick and N.-H. Chua, eds.) pp. 707-714, ElsevierBiomedical Press, New York, and Crawford, N. A., et al. (1989), Arch.Biochem. Biophys. 271:223-239), did not replace DTT.

NADP-MDH activity was proportional to the level of added DSG-1 and DSG-2at a constant thioredoxin h concentration (see FIG. 2). FIG. 2 shows theeffect of α-amylase inhibitor concentration on NADP-malate dehydrogenaseactivation by DSG-1 and DSG-2 according to the same DTT formula setforth above. Except for varying the DSG-1 or DSG-2 concentrations,conditions were identical to those previously described and shown inFIG. 1. When tested at a fixed DSG concentration, NADP-MDH showedenhanced activity with increasing thioredoxin h (as shown in FIG. 3).Except for varying the thioredoxin h concentration, conditions were asdescribed above for FIG. 1.

CM-1—the bread wheat protein that is similar to DSG proteins but has alower molecular weight—also activated NADP-MDH and not FBPase when 20 μgof CM-1 were used as shown in Table I. The results indicate thatthioredoxin h reduces a variety of α-amylase inhibitors, which, in turn,activate NADP-MDH in accordance with equations 4-6. These proteins wereineffective in enzyme activation when DTT was added in the absence ofthioredoxin.

(4) DTT_(red) + Thioredoxin h _(ox) → Thioredoxin h _(red) + DTT_(ox)(5) α-Amylase inhibitor_(ox) + Thioredoxin h _(red) → α-Amylaseinhibitor_(red) + Thioredoxin h _(ox) (6) α-Amylase inhibitor_(red) +NADP-MDH_(ox) →   (Inactive) α-Amylase inhibitor_(ox) + NADP-MDH_(red)(Active)

TABLE I Effectiveness of Thioredoxin-Reduced Trypsin Inhibitors,Thionins, and α-Amylase Inhibitors in Activating Chloroplast NADP-MalateDehydrogenase and Fructose Bisphosphatase (DTT→Thioredoxin→IndicatedProtein→Target Enzyme) No. of *ACTIVITY, nkat/mg Protein M,kDa S-SGroups NADP-MDH FBPase α-Amylase Inhibitors **DSG-2 17 5 2 0 **DSG-1 145 2 0 ‡CM-1 12 5 12  0 Trypsin Inhibitors Cystine-rich (plant) Cornkernel 12 5 5 0 Soybean Bowman-Birk 8 7 3 0 Other types Ovomucoid 28 9 20 Soybean Kunitz 20 2 2 0 Ovoinhibitor 49 14 1 0 Bovine lung (Aprotinin)7 3 Trace 2 Thionins **Purothionin-α, 6 4 1 39  **Purothionin-β 6 4Trace 5 ‡Purothionin-α 6 4 0 14  Activation of NADPH-MDH was carried outas in FIG. 1 except that the quantity of DSG or the other proteinstested was 20 μg. FBPase activation was tested using the # standard DTTassay with 1 μg of E. coli thioredoxin and 20 μg of the indicatedproteins. The above values are corrected for the limited activation seenwith E. coli thioredoxin under these conditions (see FIG. 1). *Thesevalues compare to the corresponding values of 40 and 550 obtained,respectively, with spinach chloroplast thioredoxin m(NADP-MDH) andthioredoxin f. **From Durum wheat ‡From bread wheat

EXAMPLE 2 DTNB Reduction Assays

A second test for thiol redox activity is the ability to catalyze thereduction of the sulfhydryl reagent, 2′,5′-dithiobis(2-nitrobenzoicacid) (DTNB), measured by an increase in absorbance at 412 nm. Here, theprotein assayed was reduced with NADPH via NTR and a thioredoxin. TheDTNB assay proved to be effective for the α-amylase inhibitors from bothdurum (DSG-1 and 2) and bread wheat (CM-1). When reduced by theNADP/thioredoxin system (in this case. using NTR and thioredoxin from E.coli), either DSG-1 or DSG-2 markedly enhanced the reduction of DTNB(FIG. 4). The uppermost curve in FIG. 4 represents results obtained witheither DSG-1 or -2 (NADPH→NTR→Thioredoxin DSG→DTNB). The DTNB reductionassay was carried out with 10 μg thioredoxin and 10 μg NTR and 20 μg ofDSG-1 or DSG-2. CM-1 was also effective in the DTNB reduction assay,and, as with NADP-MDH activation (Table I), was detectably more activethan the DSG proteins (See, FIG. 5, conditions were as in FIG. 4 exceptthat the DSG proteins were omitted and purothionin α, 20 μg or CM-1, 20μg was used). The results thus confirmed the enzyme activationexperiments in Example 1 and showed that the α-amylase inhibitors can bereduced physiologically by the NADP/thioredoxin system. The role of theα-amylase inhibitors in promoting the reduction of DTNB under theseconditions is summarized in equations 7-9.

EXAMPLE 3 Protein Reduction Measurements

The availability of monobromobimane (mBBr) and its adaptation for use inplant systems has given a new technique for measuring the sulfhydrylgroups of plant proteins (Crawford, N. A., et al. (1989), Arch. Biochem.Biophys. 271:223-239). When coupled with SDS-polyacrylamide gelelectrophoresis, mBBr can be used to quantitate the change in thesulfhydryl status of redox active proteins, even in complex mixtures.This technique was therefore applied to the inhibitor proteins toconfirm their capacity for reduction by thioredoxin. Here, the testprotein was reduced with thioredoxin which itself had been previouslyreduced with either DTT or NADPH and NTR. The mBBr derivative of thereduced protein was then prepared, separated from other components bySDS-polyacrylamide gel electrophoresis and its reduction state wasexamined by fluorescence. In the experiments described below,thioredoxin from E. coli was found to be effective in the reduction ofeach of the targeted proteins. Parallel experiments revealed thatthioredoxin h and calf thymus thioredoxins reduced, respectively, theproteins from seed and animal sources.

In confirmation of the enzyme activation and dye reduction experiments,DSG-1 was effectively reduced in the presence of thioredoxin. Followingincubation the proteins were derivatized with mBBr and fluorescencevisualized after SDS-polyacrylamide gel electrophoresis (FIG. 6).Reduction was much less with DTT alone and was insignificant with GSH. Asimilar requirement for thioredoxin was observed for the reduction ofCM-1 (FIG. 7) and DSG-2 (data not shown). While the thioredoxin used inFIGS. 6 and 7 was from E. coli similar results were obtained with wheatthioredoxin h. Thioredoxin was also required when DTT was replaced byNADPH and NTR (data not shown).

EXAMPLE 4 Thioredoxin-linked Reduction of Cystine-Rich Plant TrypsinInhibitors

Whereas the major soluble cystine-rich proteins of wheat seeds can actas inhibitors of exogenous α-amylases, the cystine-rich proteins of mostother seeds lack this activity, and, in certain cases, act as specificinhibitors of trypsin from animal sources. While these proteins can bereduced with strong chemical reductants such as sodium borohydride(Birk, Y. (1985), Int. J. Peptide Protein Res. 25:113-131, and Birl, Y.(1976), Meth. Enzymol. 45:695-7390), there is little evidence that theycan be reduced under physiological conditions. It was therefore ofinterest to test trypsin inhibitors for the capacity to be reduced bythioredoxin. The cystine-rich representatives tested included thesoybean Bowman-Birk and corn kernel trypsin inhibitors. The results inboth cases were positive: each inhibitor activated NADP-MDH (but notFBPase) when added in the presence of DTT-reduced thioredoxin (Table I)and each reduced DTNB in the presence of NADPH, NTR and thioredoxin(data not shown).

As found for the α-amylase inhibitors, the thioredoxin-dependentreduction of the cystine-rich trypsin inhibitors could be directlymonitored by the mBBr/SDS-polyacrylamide gel electrophoresis technique.Thus, significant reduction by DTT was observed only in the presence ofreduced thioredoxin with both the Bowman-Birk (BBTI) inhibitor (highlyfluorescent fast moving band in FIG. 6) and corn kernel (CKTI) trypsininhibitor (highly fluorescent band migrating behind thioredoxin in FIG.7).

EXAMPLE 5 Thioredoxin-linked Reduction of Other Trypsin Inhibitors andPurothionins

In view of the finding that cystine-rich trypsin inhibitors from seedscan undergo specific reduction by thioredoxin, the question arose as towhether other types of trypsin inhibitor proteins share this property.In the course of this study, several such inhibitors—soybean Kunitz,bovine lung aprotinin, egg white ovoinhibitor and ovomucoid trypsininhibitors—were tested. While the parameters tested were not asextensive as with the cystine-rich proteins described above, it wasfound that the other trypsin inhibitors also showed a capacity to bereduced specifically by thioredoxin as measured by both the enzymeactivation and mBBr/SDS-polyacrylamide gel electrophoresis methods. Aswas the case for the cystine-rich proteins described above, the trypsininhibitors tested in this phase of the study (soybean Kunitz and animaltrypsin inhibitors) activated NADP-MDH but not FBPase (Table I). Bovinelung aprotinin was an exception in that it activated FBPase moreeffectively than NADP-MDH. It might also be noted that aprotininresembles certain of the seed proteins studied here in that it shows ahigh content of cystine (ca. 10%) (Kassel, B., et al. (1965), Biochem.Biophys. Res. Commun. 20:463-468).

The fluorescence evidence for the thioredoxin-linked reduction of one ofthese proteins, the Kunitz inhibitor, is shown in FIG. 7 (highlyfluorescent slow moving band). In its reduced form, the Kunitz inhibitoralso yielded a fluorescent fast moving band. The nature of this lowermolecular mass species is not known. Its position suggests that it couldrepresent Bowman-Birk inhibitor present as a contaminant in the Kunitzpreparation (cf. FIG. 6); however, such a component was not evident inCoomassie Blue stained SDS gels. The animal inhibitors which yielded asingle fluorescent band of the expected molecular weight, also showed athioredoxin requirement for reduction (data not shown).

In confirmation of earlier results, thioredoxin-reduced purothioninconsistently activated FBPase and the type tested earlier,purothionin-α, failed to activate NADP-MDH (Table I) (Wada, K., et al.(1981), FEBS Lett. 124:237-240). However, in contrast to purothionin-αfrom bread wheat, two purothionins previously not examined (purothioninsα-1 and β from durum wheat) detectably activated NADP-MDH (Table I). Thetwo durum wheat purothionins also differed in their ability to activateFBPase. The activity differences between these purothionins wereunexpected in view of the strong similarity in their amino acidsequences (Jones, B. L., et al. (1977), Cereal Chem. 54:511-523) and intheir ability to undergo reduction by thioredoxin. A requirement forthioredoxin was observed for the reduction of purothionin (here theα-type) by the SDS-PAGE fluorescence procedure (FIG. 7).

EXAMPLE 6 Quantitation of Reduction

The above Examples demonstrate that thioredoxin reduces a variety ofproteins, including α-amylase, such as the CM and DSG inhibitors, andtrypsin inhibitors from seed as well as animal sources. While clear inthe qualitative sense, the above results give no quantitative indicationof the extent of reduction. Therefore, an experiment was conductedfollowing the protocol of Crawford, et al. (Crawford, N. A., et al.(1989), Arch. Biochem. Biophys. 271:223-239).

As shown in Table II, the extent of reduction of the seed inhibitorproteins by the E. coli NADP/thioredoxin system was time-dependent andreached, depending on the protein, 15 to 48% reduction after two hours.The results, based on fluorescence emitted by the major proteincomponent, indicate that thioredoxin acts catalytically in the reductionof the α-amylase and trypsin inhibitors. The ratio of protein reducedafter two hours to thioredoxin added was greater than one for both themost highly reduced protein (soybean Bowman-Birk trypsin inhibitor) andthe least reduced protein (corn kernel trypsin inhibitor)—i.e.,respective ratios of 7 and 2 after a two-hour reduction period. Itshould be noted that the values in Table II were obtained under standardassay conditions and no attempt was made to optimize reduction bymodifying those conditions.

TABLE II Extent of Reduction of Seed Proteins by the NADP/ThioredoxinSystem Using the mBBr/SDS- Polyacrylamide Gel Electrophoresis Procedure% Reduction After Protein 20 min 120 min Purothionin-β 15 32 DSG-1 22 38Corn kernel trypsin  3 15 inhibitor Bowman-Birk trypsin 25 48 inhibitorKunitz trypsin inhibitor 14 22 The following concentrations of proteinswere used (nmoles): thioredoxin, 0.08; NTR, 0.01; purothionin-β, 1.7;DSG-1, 0.7; corn kernel trypsin inhibitor, 1.0; # Bowman-Birk trypsininhibitor, 1.3; and Kunitz trypsin inhibitor, 0.5. Except for theindicated time difference, other conditions were as in FIG. 6.

EXAMPLE 7 E. coli Glutaredoxin as Reductant

Bacteria and animals are known to contain a thiol redox protein,glutaredoxin, that can replace thioredoxin in reactions such asribonucleotide reduction (Holmgren, A. (1985), Annu. Rev. Biochem.54:237-271). Glutaredoxin is reduced as shown in equations 10 and 11.

So far there is no evidence that glutaredoxin interacts with proteinsfrom higher plants. This ability was tested, using glutaredoxin from E.coli and the seed proteins currently under study. Reduction activity wasmonitored by the mBBr/SDS polyacrylamide gel electrophoresis procedurecoupled with densitometric scanning. It was observed that, under theconditions developed for FIGS. 6 and 7, glutaredoxin could effectivelyreplace thioredoxin in some, but not all cases. Thus, glutaredoxin wasfound to be active in the reduction of the following (the numbersindicate the percentage reduction relative to E. coli thioredoxin):DSG-1 and CM-1 α-amylase inhibitors (147 and 210%, respectively); cornkernel trypsin inhibitor (424%); and purothionin (82, 133, and 120% forthe α, α1 and β forms, respectively). Glutaredoxin was ineffective inthe reduction of the DSG-2 α-amylase inhibitor and the soybeanBowman-Birk and Kunitz trypsin inhibitors. The trypsin inhibitors fromanimal sources also showed a mixed response to glutaredoxin. Egg whiteovoinhibitor was effectively reduced (55% reduction relative to E. colithioredoxin) whereas egg white ovomucoid inhibitor and bovine lungaprotinin were not affected. Significantly, as previously reported(Wolosiuk, R. A., et al. (1977), Nature 266:565-567), glutaredoxinfailed to replace thioredoxin as the immediate reductant in theactivation of thioredoxin-linked enzymes of chloroplasts, FBPase andNADP-MDH (data not shown).

The above Examples demonstrate that some of the enzyme inhibitorproteins tested can be reduced by glutaredoxin as well as thioredoxin.Those specific for thioredoxin include an α-amylase inhibitor (DSG-2),and several trypsin inhibitors (Kunitz, Bowman-Birk, aprotinin, andovomucoid inhibitor). Those proteins that were reduced by eitherthioredoxin or glutaredoxin include the purothionins, two α-amylaseinhibitors (DSG-1, CM-1), a cystine-rich trypsin inhibitor from plants(corn kernel inhibitor), and a trypsin inhibitor from animals (egg whiteovoinhibitor). These results raise the question of whether glutaredoxinoccurs in plants. Glutaredoxin was reported to be present in a greenalga (Tsang, M. L.-S. (1981), Plant Physiol. 68:1098-1104) but not inhigher plants.

Although the activities of the NADP-MDH and FBPase target enzymes shownin Table I are low relative to those seen following activation by thephysiological chloroplast proteins (thioredoxin m or f), the valuesshown were found repeatedly and therefore are considered to be real. Itseems possible that the enzyme specificity shown by the inhibitorproteins, although not relevant physiologically, reflects a particularstructure achieved on reduction. It remains to be seen whether such areduced structure is related to function within the seed or animal cell.

The physiological consequence of the thioredoxin (or glutaredoxin)linked reduction event is of considerable interest as the function ofthe targeted proteins is unclear. The present results offer a newpossibility. The finding that thioredoxin reduces a wide variety ofinhibitor proteins under physiological conditions suggests that, in theabsence of compartmental barriers, reduction can take place within thecell.

EXAMPLE 8 Inactivation of Soybean Trypsin Inhibitor in Soybean Meal

The goal of this Example is to inactivate the Bowman-Birk and Kunitztrypsin inhibitors of soybeans, The following protocol applies to animalfeed preparations. To 10 g of soybean meal are added 0.2 μg thioredoxin,0.1 μg NADP-thioredoxin reductase and 500 nanomoles NADPH together with1 M Tris-HCl buffer, pH 7.9, to give 5.25 ml of 30 mM Tris-HCl. Theabove mixture is allowed to sit for about 30 min. at room temperature.Direct reduction of the soybean trypsin inhibitor is determined usingthe mBBr fluorescent labeling/SDS-polyacrylamide gel electrophoresismethod previously described (Kobrehel, K., et al. (1991), J. Biol. Chem.266:16135-16140). An analysis of the ability of the treated flour fortrypsin activity is made using modifications of the insulin and BAEE(Na-benzoyl-L-arginine ethyl ester) assays (Schoellmann, G., et al.(1963), Biochemistry 252:1963; Gonias, S. L., et al. (1983), J. Biol.Chem. 258:14682). From this analysis it is determined that soybean mealso treated with the NADP/thioredoxin system does not inhibit trypsin.

EXAMPLE 9 Inactivation of α-Amylase Inhibitors in Cereals

To 10 g of barley malt are added 0.2 μg thioredoxin, 0.1 μgNADP-thioredoxin reductase and 500 nanomoles NADPH together with 1 MTris-HCl buffer, pH 7.9, to give 5.25 ml of 30 mM Tris-HCl. The abovemixture is allowed to sit for about 30 min. at room temperature. Directreduction of the α-amylase inhibitors is determined using the mBBrfluorescent labeling/SDS-polyacrylamide gel electrophoresis methodpreviously described (Kobrehel, K., et al. (1991), J. Biol. Chem.266:16135-16140). α-Amylase activity is monitored by following therelease of maltose from starch (Bernfeld, P. (1955), Methods in Enzymol.1:149). From this analysis it is determined that barley so treated withthe NADP/thioredoxin system does not inhibit α-amylase.

Reduction of Cereal Proteins Materials and Methods

Plant Material

Seeds and semolina of durum wheat (Triticum durum, Desf. cv. Monroe)were kind gifts of Dr. K. Kahn.

Germination of Wheat Seeds

Twenty to 30 seeds were placed in a plastic Petri dish on three layersof Whatman #1 filter paper moistened with 5 ml of deionized water.Germination was carried out for up to 4 days at room temperature in adark chamber.

Reagents/Fine Chemicals

Biochemicals and lyophilized coupling enzymes were obtained from SigmaChemical Co. (St. Louis, Mo.). E. coli thioredoxin and NTR werepurchased from American Diagnostica, Inc. (Greenwich, Conn.). Wheatthioredoxin h and NTR were isolated from germ, following the proceduresdeveloped for spinach leaves (Florencio, F. J., et al. (1988), Arch.Biochem. Biophys. 266:496-507). E. coli glutaredoxin was a kind gift ofProfessor A. Holmgren. Reagents for SDS-polyacrylamide gelelectrophoresis were purchased from Bio-Rad Laboratories (Richmond,Calif.). Monobromobimane (mBBr) or Thiolite was obtained from CalbiochemCo. (San Diego, Calif.). Aluminum lactate and methyl green were productsof Fluka Chemicals Co. (Buchs, Switzerland).

Gliadins and Glutenins

For isolation of insoluble storage proteins, semolina (0.2 g) wasextracted sequentially with 1 ml of the following solutions for theindicated times at 25° C.: (1) 50 mM Tris-HCl, pH 7.5 (20 min); (2) 70%ethanol (2 hr); and (3) 0.1 M acetic acid (2 hr). During extraction,samples were placed on an electrical rotator and, in addition,occasionally agitated with a vortex mixer. After extraction with eachsolvent, samples were centrifuged (12,000 rpm for 5 min.) in anEppendorf microfuge and, supernatant fractions were saved for analysis.In between each extraction, pellets were washed with 1 ml of water,collected by centrifugation as before and the supernatant wash fractionswere discarded. By convention, the fractions are designated: (1)albumin/globulin; (2) gliadin; and (3) glutenin.

In vitro mBBr Labelling of Proteins

Reactions were carried out in 100 mM Tris-HCl buffer, pH 7.9. Asindicated, 0.7 μg NTR and 1 μg thioredoxin (both from E. coli unlessspecified otherwise) were added to 70 μl of this buffer containing 1 mMNADPH and 10 μg of target protein. When thioredoxin was reduced bydithiothreitol (DTT), NADPH and NTR were omitted and DTT was added to0.5 mM. Assays with reduced glutathione were performed similarly, but ata final concentration of 1 mM. After incubation for 20 min, 100 nmolesof mBBr were added and the reaction was continued for another 15 min. Tostop the reaction and derivatize excess mBBr, 10 μl of 10% SDS and 10 μlof 100 mM β-mercaptoethanol were added and the samples were then appliedto the gels. For reduction by glutaredoxin, the thioredoxin and NTR werereplaced by 1 μg E. coli glutaredoxin, 1.4 μg glutathione reductase(purified from spinach leaves) and 1.5 mM NADPH.

In vivo mBBr Labelling of Proteins

At the indicated times, the dry seeds or germinating seedlings (selectedon the basis of similar radical length) were removed from the Petri dishand their embryos or germinated axes were removed. Five endosperms fromeach lot were weighed and then ground in liquid N₂ with a mortal andpestle. One ml of 2.0 mM mBBr in 100 mM Tris-HCl, pH 7.9, buffer wasadded just as the last trace of liquid N₂ disappeared. The thawedmixture was then ground for another minute and transferred to amicrofuge tube. The volume of the suspension was adjusted to 1 ml withthe appropriate mBBr or buffer solution. Protein fractions ofalbumin/globulin, gliadin and glutenin were extracted from endosperm ofgerminated seedlings as described above. The extracted protein fractionswere stored at −20° C. until use. A buffer control was included for eachtime point.

SDS-Polyacrylamide Gel Electrophoresis

SDS-polyacrylamide electrophoresis of the mBBr-derivatized samples wasperformed in 15% gels at pH 8.5 as described by Laemmli, U. K. (1970),Nature 227:680-685. Gels of 1.5 mm thickness were developed for 16 hr.at a constant current of 9 mA.

Native Gel Electrophoresis

To resolve the different types of gliadins, native polyacrylamide gelelectrophoresis was performed in 6% gels (a procedure designed toseparate gliadins into the four types) as described by Bushuk andZillman (Bushuk, W., et al. (1978), Can. J. Plant Sci. 58:505-515) andmodified for vertical slab gels by Sapirstein and Bushuk (Sapirstein, H.D., et al. (1985), Cereal Chem. 62:372-377). A gel solution. in 100 mlfinal volume contained 6.0 g acrylamide, 0.3 g bisacrylamide, 0.024 gascorbic acid, 0.2 mg ferrous sulfate heptahydrate and 0.25 g aluminumlactate. The pH was adjusted to 3.1 with lactic acid. The gel solutionwas degassed for 2 hr. on ice and then 0.5 ml of 3% hydrogen peroxidewas added as a polymerization catalyst. The running buffer, alsoadjusted to pH 3.1 with lactic acid, contained 0.5 g aluminum lactateper liter. Duration of electrophoresis was approximately 4 hr., with aconstant current of 50 mA. Electrophoresis was terminated when thesolvent front, marked with methyl green tracking dye, migrated to about1 cm from the end of the gel.

mBBr Removal/Fluorescence Photography

Following electrophoresis, gels were placed in 12% (w/v) trichloroaceticacid and soaked for 4 to 6 hr. with one change of solution to fix theproteins; gels were then transferred to a solution of 40% methanol/10%acetic acid for 8 to 10 hr. to remove excess mBBr. The fluorescence ofmBBr, both free and protein bound, was visualized by placing gels on alight box fitted with an ultraviolet light source (365 nm). Followingremoval of the excess (free) mBBr, gels were photographed with PolaroidPositive/Negative Landfilm, type 55, through a yellow Wratten gelatinfilter No. 8 (cutoff=460 nm) (exposure time ranged from 25 to 60 sec atf4.5) (Crawford, N. A., et al. (1989), Arch. Biochem. Biophys.271:223-239).

Protein Staining/Destaining/Photography

SDS-gels were stained with Coomassie Brilliant Blue R-250 in 40%methanol/10% acetic acid for 1 to 2 hr. and destained overnight asdescribed before (Crawford, N. A., et al. (1989), Arch. Biochem.Biophys. 271:223-239). Aluminum lactate native gels were stainedovernight in a filtered solution containing 0.1 g Coomassie BrilliantBlue R-250 (dissolved in 10 ml 95% ethanol) in 240 ml 12%trichloroacetic acid. Gels were destained overnight in 12%trichloroacetic acid (Bushuk, W., et al. (1978), Can. J. Plant Sci.58:505-515, and Sapirstein, H. D., et al. (1985), Cereal Chem.62:372-377).

Protein stained gels were photographed with Polaroid type 55 film toproduce prints and negatives. Prints were used to determine bandmigration distances and loading efficiency.

The Polaroid negatives of fluorescent gels and prints of wet proteinstained gels were scanned with a laser densitometer (Pharmacia-LKBUltroScan XL). Fluorescence was quantified by evaluating peak areasafter integration with GelScan XL software.

Enzyme Assays

The following activities were determined in crude extracts withpreviously described methods: hexokinase (Baldus, B., et al. (1981),Phytochem. 20:1811-1814), glucose-6-phosphate dehydrogenase,6-phosphogluconate dehydrogenase (Schnarrenberger, C., et al. (1973),Arch. Biochem. Biophys. 154:438-448), glutathione reductase, NTR andthioredoxin h (Florencio, F. J., et al. (1988), Arch. Biochem. Biophys.266:496-507).

Protein Determination

Protein concentrations were determined by the Bradford method (Bradford,M. (1976) Anal. Biochem. 72:248-256), with Bio-Rad reagent and bovineserum albumin as a standard.

Subunit Molecular Weight Determination

The subunit molecular weight of gliadins and glutenins was estimated onSDS-PAGE gels by using two sets of molecular weight standards (kDa). Thefirst set consisted of BSA (66), ovalbumin (45), soybean trypsininhibitor (20.1), myoglobin (17), cytochrome c (12.4) and aprotinin(6.5). The other set was the BioRad Prestained Low SDS-PAGE standards:phosphorylase b (110), BSA (84), ovalbumin (47), carbonic anhydrase(33), soybean trypsin inhibitor (24) and lysozyme (16).

EXAMPLE 10 Reduction of Gliadins

As a result of the pioneering contributions of Osborne and coworkers acentury ago, seed proteins can be fractionated on the basis of theirsolubility in aqueous and organic solvents (20). In the case of wheat,preparations of endosperm (flour or semolina) are historicallysequentially extracted with four solutions to yield the indicatedprotein fraction: (i) water, albumins; (ii) salt water, globulins; (iii)ethanol/water, gliadins; and (iv) acetic acid/water, glutenins. A widebody of evidence has shown that different proteins are enriched in eachfraction. For example, the albumin and globulin fractions containnumerous enzymes, and the gliadin and glutenin fractions are in thestorage proteins required for germination.

Examples 1, 4 and 5 above describe a number of water soluble seedproteins (albumins/globulins, e.g., α-amylase inhibitors, cystine-richtrypsin inhibitors, other trypsin inhibitors and thionines) that arereduced by the NADP/thioredoxin system, derived either from the seeditself or E. coli. The ability of the system to reduce insoluble storageproteins from wheat seeds, viz., representatives of the gliadin andglutenin fractions, is described below. Following incubation with theindicated additions, the gliadin proteins were derivatized with mBBr andfluorescence was visualized after SDS-polyacrylamide gelelectrophoresis. The lanes in FIG. 8 were as follows: 1. Control: noaddition. 2. GSH/GR/NADPH: reduced glutathione, glutathione reductase(from spinach leaves) and NADPH. 3. NGS: NADPH, reduced glutathione,glutathione reductase (from spinach leaves) and glutaredoxin (from E.coli). 4. NTS: NADPH, NTR, and thioredoxin (both proteins from E. coli).5. MET/T(Ec): β-mercaptoethanol and thioredoxin (E. coli). 6. DTT. 7.DTT/T(Ec): DTT and thioredoxin (E. coli). 8. DTT/T(W): Same as 7 exceptwith wheat thioredoxin h. 9. NGS,-Gliadin: same as 3 except without thegliadin protein fraction. 10. NTS,-Gliadin: same as 4 except without thegliadin protein fraction. Based on its reactivity with mBBr, the gliadinfraction was extensively reduced by thioredoxin (FIG. 8). The majormembers undergoing reduction showed a Mr ranging from 25 to 45 kDa. Asseen in Examples 1, 4 and 5 with the seed α-amylase and trypsininhibitor proteins, the gliadins were reduced by either native h or E.coli type thioredoxin (both homogeneous); NADPH (and NTR) or DTT couldserve as the reductant for thioredoxin. Much less extensive reductionwas observed with glutathione and glutaredoxin—a protein able to replacethioredoxin in certain E. coli and mammalian enzyme systems, but notknown to occur in higher plants.

The gliadin fraction is made up of four different protein types,designated α, β, γ and ω, which can be separated by nativepolyacrylamide gel electrophoresis under acidic conditions (Bushuk, W.,et al. (1978), Can. J. Plant Sci. 58:505-515; Kasarda, D. D., et al.(1976), Adv. Cer. Sci. Tech. 1:158-236; Sapirstein, H. D., et al.(1985), Cereal Chem. 62:372-377; and Tatham, A. S., et al. (1990), Adv.Cer. Sci. Tech. 10:1-78). Except for the ω gliadins, each speciescontains cystine (S—S) groups and thus has the potential for reductionby thioredoxin. In this study, following incubation with the indicatedadditions, proteins were derivatized with mBBr, and fluorescence wasvisualized after acidic-polyacrylamide gel electrophoresis. The lanes inFIG. 9 were as follows: 1. Control: no addition. 2. GSH: reducedglutathione. 3. GSH/GR/NADPH: reduced glutathione, glutathione reductase(from spinach leaves) and NADPH. 4. NGS: NADPH, reduced glutathione,glutathione reductase (from spinach leaves) and glutaredoxin (from E.coli). 5. NGS+NTS: combination of 4 and 6. 6. NTS: NADPH, NTR, andthioredoxin (both proteins from E. coli). 7. MET/T(Ec):β-mercaptoethanol and thioredoxin (E. coli). 8. DTT/T(Ec): DTT andthioredoxin (E. coli). 9. NTS(-T): same as 6 except without thioredoxin.10. NGS+NTS,-Gliadin: same as 5 except without the gliadin fraction.

When the thioredoxin-reduced gliadin fraction was subjected to nativegel electrophoresis, the proteins found to be most specifically reducedby thioredoxin were recovered in the a fraction (See, FIG. 9). There wasactive reduction of the β and γ gliadins, but as evident from thedensitometer results summarized in Table III, the reduction within thesegroups was nonspecific, i.e., relatively high levels of reduction werealso achieved with glutathione and glutaredoxin. There was especiallystrong reduction of the γ gliadins by DTT-reduced thioredoxin (FIG. 9).As anticipated, there was no reduction of the ω gliadins. The evidenceindicates that thioredoxin (either native h or E. coli) specificallyreduces certain of the gliadins, especially the α type.

EXAMPLE 11 Reduction of Glutenins

The remaining group of seed proteins to be tested for a response tothioredoxin—the glutenins—while the least water soluble, are perhaps ofgreatest interest. The glutenins have attracted attention over the yearsbecause of their importance for the cooking quality of flour andsemolina (MacRitchie, F., et al. (1990), Adv. Cer. Sci. Tech.10:79-145). Testing the capability of thioredoxin to reduce the proteinsof this group was, therefore, a primary goal of the currentinvestigation.

TABLE III Reductant Specificity of the Different Types of GliadinsGliadin, % Relative Reduction Reductant α β γ Aggregate* None 22.4 30.424.3 29.2 Glutathione 36.4 68.1 60.6 60.1 Glutaredoxin 43.5 83.3 79.761.5 Thioredoxin 100.0 100.0 100.0 100.0 The area under α, β, γ andaggregate peaks following reduction by the NADP/thioredoxin system were:4.33, # 8.60, 5.67 and 0.74 Absorbance units times millimeters,respectively. These combined areas were about 65% of those observed whenthioredoxin was reduced by DTT. Reaction conditions as in FIG. 9.*Proteins not entering the gel

As seen in FIG. 10 (treatments were as in Example 10, FIG. 8), severalglutenins were reduced specifically by thioredoxin. The most extensivereduction was observed in the low molecular mass range (30-55 kDa). Thereduction observed in the higher molecular mass range was lesspronounced, but still obvious—especially in the 100 kDa region andabove. Though not shown reduction may also occur in the 130 kDa range.Like the gliadins, certain of the glutenins were appreciably reduced byglutathione and glutaredoxin. However, in all cases, reduction wasgreater with thioredoxin and, in some cases, specific to thioredoxin(Table IV, note proteins in the 30-40 and 60-110 kDa range). As observedwith the other wheat proteins tested, both the native h anal E. colithioredoxins were active and could be reduced with either NADPH and thecorresponding NTR or with DTT. Thus as found for the gliadins, certainglutenins were reduced in vitro specifically by thioredoxin, whereasothers were also reduced, albeit less effectively, by glutathione andglutaredoxin.

TABLE IV Reductant Specificity of Glutenins Reaction conditions as inFIG. 3. Glutenin, % Relative Reduction* Reductant 60-110 kDa 40-60 kDa30-40 kDa None 8 23 16 Glutathione 31 51 29 Glutaredoxin 50 72 40Thioredoxin* 100 100 100 *Area under the three molecular weight classes(from high to low) following reduction by the NADP/thioredoxin systemwere: 1.5, 5.67 and 5.04 Absorbance units times millimeters,respectively.

EXAMPLE 12 In vivo Reduction Experiments

The above Example demonstrates that thioredoxin specifically reducescomponents of the wheat gliadin and glutenin fractions when tested invitro. The results, however, provide no indication as to whether theseproteins are reduced in vivo during germination—a question that, to ourknowledge, had not been previously addressed (Shutov, A. D., et al.(1987), Phytochem. 26:1551-1566).

To answer this question, we applied the mBBr/SDS-PAGE technique wasapplied to monitor the reduction status of proteins in the germinatingseed. We observed that reduction of components in the Osborne fractionsincreased progressively with time and reached a peak after 2 to 3 daysgermination (FIG. 11). The observed increase in reduction ranged from2-fold with the gliadins, to 3-fold with the albumin/globulins and5-fold with the glutenins. The results suggest that, whilerepresentatives of the major wheat protein groups were reduced duringgermination, the net redox change was greatest with the glutenins.

Although providing new evidence that the seed storage proteins undergoreduction during germination, the results of FIG. 11 give no indicationas to how reduction is accomplished, i.e., by glutathione orthioredoxin. To gain information on this point, the in vivo reductionlevels of the principal thioredoxin-linked gliadins (30-50 kDa) andglutenins (30-40, 40-60 kDa) was compared with the reduction determinedfrom in vitro measurements (cf. FIG. 8 and Table IV). For this purpose,the ratio of fluorescence to Coomassie stained protein observed in vivoduring germination and in vitro with the appropriate enzyme reductionsystem was calculated. The results shown in FIG. 12 (principalthioredoxin linked gliadins were those in the Mr range from 25 to 45kDa, see FIG. 8, and glutenins were those in the Mr range from 30 to 60kDa, see FIG. 10) suggest that, while glutathione could account for asignificant part of the in vivo reduction of the gliadin fraction (up to90%), this was not the case with the glutenins whose reduction seemed torequire thioredoxin. The level of reduction that could be ascribed toglutathione (or glutaredoxin) was insufficient to account for the levelsof reduced glutenin measured in the germinating seed.

EXAMPLE 13 Enzyme Measurements

The source of NADPH needed for the NTR linked reduction of thioredoxin hwas also investigated. Semolina was analyzed for enzymes that functionin the generation of NADPH in other systems, notably dehydrogenases ofthe oxidative phosphate pathway. The results summarized in Table Vconfirm earlier evidence that endosperm extracts contain the enzymesneeded to generate NADPH from glucose via this pathway: hexokinase,glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase(Tatham, A. S., et al. (1990), Adv. Cer. Sci. Tech. 10:1-78). It isnoteworthy that the glucose 6-phosphate dehydrogenase activity seen inTable V was insensitive to reduced thioredoxin (data not shown). In thisrespect the endosperm enzyme resembles its cytosolic rather than itschloroplast counterpart from leaves (Fickenscher, K., et al. (1986),Arch. Biochem. Biophys. 247:393-402; Buchanan, B. B. (1991), Arch.Biochem. Biophys. 288:1-9; Scheibe, R., et al. (1990), Arch. Biochem.Biophys. 274:290-297).

As anticipated from earlier results with flour (Johnson, T. C., et al.(1987), Planta 171:321-331; Suske, G., et al. (1979), Z. Naturforsch. C34:214-221), semolina also contained thioredoxin h and NTR (Table V).Interestingly, based on activity measurements, NTR appeared to be arate-limiting component in preparations from the cultivar examined.

TABLE V Activities of Enzymes Effecting the Reduction of Thioredoxin hinSemolina (Glucose→Glu-6-P→6-P-Gluconate→NADP→Thioredoxin h) ActivityProtein (nkat/mg protein) Hexokinase 0.28 Glucose-6-P dehydrogenase 0.456-P-Gluconate dehydrogenase 0.39 NTR 0.06 Thioredoxin h 0.35

The present results suggest that thioredoxin h functions as a signal toenhance metabolic processes associated with the germination of wheatseeds. Following its reduction by NTR and NADPH (generated via theoxidative pentose phosphate pathway), thioredoxin h appears to functionnot only in the activation of enzymes, but also in the mobilization ofstorage proteins.

EXAMPLE 14 Improvement of Dough Quality

Dough quality was improved by reducing the flour proteins using theNADP/thioredoxin system. Reduced thioredoxin specifically breakssulfur-sulfur bonds that cross-link different parts of a protein andstabilize its folded shape. When these cross-links are cut the proteincan unfold and link up with other proteins in bread, creating aninterlocking lattice that forms the elastic network of dough. The doughrises because the network traps carbon dioxide produced by yeast in thefermenting process. It is proposed that the reduced thioredoxinactivated the gliadins and glutenins in flour letting them recombine ina way that strengthened the dough (FIG. 13). Reduced thioredoxinstrengthened the protein network formed during dough making. For thesetests, namely those shown in FIG. 14(c) and FIG. 15(d) (using 10 gm ofeither intermediate quality wheat flour obtained from a local miller inMontpellier, France (FIG. 14), or poor quality wheat also obtained froma local miller in Montpellier, France (FIG. 15), this poor quality wheatbeing mainly of the Apollo cultivar), 0.2 μg E. coli thioredoxin, 0.1 μgE. coli NADP-thioredoxin reductase and 500 nanomoles NADPH were addedtogether with 1 M Tris-HCl, pH 7.9 buffer to give 5.25 ml of a 30 mMTris-HCl enzyme system mixture. The reaction was carried out by mixingthe enzyme system mixture with the 10 gm of the flour in amicro-farinograph at 30° C. As seen in FIGS. 14 and 15, the resultingfarinograph measurements showed a strengthening of the dough by theadded NADP/thioredoxin system. With a flour of poor quality, as in FIG.15(d), the farinograph reading was stable for at least 4 min. after thedough was formed in the presence of the reduction system, whereas thereading dropped immediately after dough formation in the control withoutthis addition (see FIG. 15(a)). The improving effect was persistent andwas maintained throughout the run. Expressed another way, themicro-farinograph reading is 375 Brabender units, 7 min. after doughformation with the poor quality wheat control (no added enzyme system)versus 450 Brabender units for the same poor quality wheat treated withcomponents of the NADP/thioredoxin system (NADPH, thioredoxin andNADP-thioredoxin reductase).

Another farinograph study was carried out as above with 10 gm of Apolloflour only the concentration of NADPH was 500 μmoles instead ofnanomoles. As shown in the farinograph measurements in FIG. 16 thioamount of NADPH also resulted in a definite improvement in the qualityof the dough.

Higher farinograph measurements of dough correspond to improved doughstrength and improved baked good characteristics such as better crumbquality, improved texture and higher loaf volume. Also, based on in vivoanalyses with the isolated proteins, the native wheat seedNADP/thioredoxin system will also be effective in strengthening thedough.

For purposes of baking and other aspects of this invention, ranges ofabout 0.1 to 3.0 μg of a thioredoxin (preferably E. coli or thioredoxinh) and from about 0.1 to 2.0 μg reductase and about 30 to 500 nanomolesof NADPH are added for about every 10 gm of flour. The optimal levels ofthioredoxin and reductase depend on flour quality. In general, thehigher the flour quality, the higher the level of thioredoxin andreductase required. Thioredoxin can also be reduced by lipoic acidinstead of by the NADPH/NADP-thioredoxin reductase reduction system. Theother dough ingredients such as milk or water are then added. However,the liquid may first be added to the NTR/thioredoxin system and thenadded to the flour. It is preferred that yeast for purposes of leaveningbe added after the reduced thioredoxin has had a chance to reduce thestorage proteins. The dough is then treated as a regular dough proofed,shaped, etc. and baked.

NADPH can be replaced in this Example as well as in the followingExamples with an NADPH generator such as one consisting of 100 μMglucose 6-phosphate, 100 μM NADP and 0.05 units (0.2 μgram) glucose6-phosphate dehydrogenase from a source such as yeast. The NADPHgenerator is added together with thioredoxin and NADP-thioredoxinreductase at the start of the dough making process.

FIG. 17(c) shows the higher farinograph measurement obtained when 10 gmof Apollo cultivar (CV) wheat are reacted with 20 μl NADP (25 mM), 20 μlG6P (25 mM), 0.25 μg G6PDase, 0.1 μg NTR and 0.2 μg thioredoxin hcontained in 4.25 ml H₂O and 0.90 ml Tris-HCl (30 mM, pH 7.9). FIG.17(b) shows that a higher farinograph measurement is also obtained when10 gm of Apollo wheat are reacted with the same reaction mixture as themixture resulting in FIG. 17(c) but without any NTR or thioredoxin.

EXAMPLE 15 Wheat Bread Baking Studies

The baking tests were carried out by using a computer monitoredPANASONIC baking apparatus.

Composition of Bread

Control: Flour*: 200 gm (dry) Water: 70% hydratation Salt (NaCl): 5.3 gYeast: 4.8 g (Saccharomyces cerevisiae, SafInstant) (dry yeast powder)*Flour samples were obtained from pure bread wheat cultivars havingcontrasting baking quality (including animal feed grade and other gradeshaving from poor to good baking quality).

Assays

The dough for the assays contained all the components of the controlplus as indicated varying amounts of the NADP Thioredoxin System (NTS)and/or the NADP generating System.

Experimental Conditions

Flour and salt are weighed and mixed

The volume of water needed to reach a hydratation of 70% was put intothe baking pan.

The mixture of flour and salt was added to the water and the bakingprogram monitored by the computer was started. The complete programlasted 3 hrs 9 min and 7 secs.

In the case of the assays, enzyme system components are added to thewater before the addition of the flour-salt mixture.

Yeast was added automatically after mixing for 20 min and 3 secs.

The program monitoring the Panasonic apparatus was:

Mixing Segments Duration Conditions Heating Mixing 00:00:03 T1 offMixing 00:05:00 T2 off Mixing 00:05:00 T1 off Rest 00:10:00 T0 offMixing 00:17:00 T2 off Mixing 00:07:00 T1 off Rest 00:30:00 T0  to reach32° C. Mixing 00:00:04 T1  32° C. Rest 01:15:00 T0  32° C. Baking00:14:00 T0 to reach 180° C. Baking 00:26:00 T0 180° C. MixingConditions: T0 = no mixing (motor at rest) T1 = normal mixing T2 =alternately 3 second mixing, 3 second rest

Bread loaf volume was determined at the end of the baking, when breadloaves reached room temperature.

Cultivar THESEE Assay

The french wheat cultivar Thesee is classified as having goodbreadmaking quality. Table VI below sets forth the results of the assay.

TABLE VI Loaf Volume NADPH NTR Th Relative (μmoles) (μg) (μg) (cm3)Units Control 0 0 0 1690 100 Samples 6.0 30 60 1810 107 6.0 30 0 1725102 6.0 0 60 1720 102 6.0 0 0 1550 92 0 30 60 1800 107 *NADPH 30 60 162096 Generating syst. *NADPH 30 60 1630 96 Generating syst. plus ATP,glucose NTR and 6.0 9.4 20 1750 104 Th from yeast *Composition of theNADPH generating system, ATP and glucose. Volume Added NADP, 25 mMolar700 μl (17.5 μmoles) Glucose-6-phosphate, 25 mMolar 700 μl (17.5 μmoles)Glucose-6-phosphate dehydrogenase (50 μg/ml) 175 μl (8.75 μg) ATP, 25mMolar 700 μl (17.5 μmoles) Glucose, 25 mMolar 700 μl (17.5 μmoles)

As shown in Table VI, an increased loaf volume was obtained when thecomplete NTS at concentrations of 6.0 μmoles NADPH, 30 μg NTR and 60 μgTh was used to bake loaves from 200 g of Thesee flour with the amountsand conditions described above in this Example. Unless otherwise stated,the NTR and thioredoxon (th) were from E. coli. No similar increaseoccurred when the generating system was used or when either NTR or Thwere omitted. Also no significant effect on loaf volume occurred whenamounts of the components in the system were about half or less thanhalf of the amounts of above.

Cultivar APOLLO Assay

This French wheat cultivar is classified as having poor breadmakingquality. The NTR and thioredoxin used in this assay were from E. coli.Table VII below sets forth the results of this assay using 200 gm ofApollo flour. Again unless otherwise stated the amounts and conditionsare those described above at the beginning of the Example.

TABLE VII Loaf Volume NADPH NTR Th Relative (μmoles) (μg) (μg) (cm3)Units Control 0 0 0 1400 100 Samples 6.0 30 60 1475 105 *NADPH 30 601530 109 Generating syst. plus ATP, glucose *NADPH 0 0 1430 102Generating syst. plus ATP, glucose *NADPH 6 0 1430 102 Generating syst.*NADPH 6 7 1440 103 Generating syst. *The composition of the generatingsystem, ATP and glucose is as in Table VI.

Cultivar ARBON Assay

The French wheat cultivar Arbon is used for feed and is classified asnon suitable for breadmaking. Tables VIII and IX below show that animproved bread loaf volume can be obtained from Arbon using the NTS orNADPH and NTR with the dough components and conditions described at thebeginning of the Example. The amounts of NTR, thioredoxin, NADPH and theNADPH generating system components used in the assay are set forth inTables VIII and IX. The improvement in Arbon bread quality using thecomplete NTS as set forth in Table IX is also clearly seen in thephotographs shown in FIGS. 18-22 and 23(a).

TABLE VIII NADPH NTR Th Loaf Volume (μmoles) (μg) (μg) (cm3) Control 0   0    0 1350 Samples 0.1-0.6 3-4 3-4 up to 20% higher than thecontrol >2.0 >20 >20 less than the control

TABLE IX Loaf Volume Relative Treatment (cm3) Units Complete NTS 1650122 minus Thioredoxin 1690 125 minus NTR 1520 113 minus Thioredoxin, NTR1540 114 minus NADPH 1440 107 minus NADPH, plus *NADPH 1560 116generating system minus NTS (control) 1350 100 NADPH, 0.6 μmolesThioredoxin, 3.5 μg NTR, 3 μg *Generating System: 3.5 μmoles NADP 3.5μmoles glucose-6-phosphate 1.75 μg glucose-6-phosphate dehydrogenase

EXAMPLE 16 Triticale Bread Baking Study

Triticale is a wheat/rye hybrid and is generally used for chicken feed.It is more nutritious than wheat but is not generally consideredappropriate for breadmaking, especially in the more developed nations.The effect of the NTS system and variations thereof on loaves baked fromTriticale flour was consequently studied. Unless otherwise stated, thebaking conditions and dough ingredient were as described for wheat flourin Example 15. As shown in Table X there is an improvement in loafvolume when the triticale dough contained thioredoxin, NTR and the NADPHgenerating system in the amounts set forth in that Table. However, nocorresponding improvement was seen when the NTS (i.e., thioredoxin, NTRand NADPH) was used. FIG. 24 shows that an improvement in the texture ofthe bread also occurred when NTR, Th and the NADPH generating system asset forth in Table X were used. The loaf on the right in FIG. 24 is thecontrol.

TABLE X Effect of the NADP/Thioredoxin System (NTS) on Loaves Baked fromTriticale Flour (cv. Juan) Loaf Volume Relative Treatment (cm3) UnitsComplete NTS 1230  94 minus NTS (control) 1310 100 minus NADPH, plus*NADPH 1390 106 generating system NADPH, 0.6 μmoles Thioredoxin, 3.5 μgNTR, 3.0 μg Generating System: 4.5 μmoles NADP 4.5 μmolesglucose-6-phosphate 4.5 μg glucose-6-phosphate dehydrogenase

EXAMPLE 17

The effect of the NADPH/thioredoxin system on flour from sorghum, cornand rice was also determined. The baking conditions were as describedfor wheat flour in Example 15. The amounts of the components of the NTSas used in this assay were as follows: 8 μmoles NADPH, 40.5 μg NTR and54 μg thioredoxin. Both the thioredoxin and NTR were from E. coli. Theresults of this assay are shown in FIG. 25 and also in FIG. 23(b). Asshown in these figures the breads containing the NTS, especially cornand sorghum exhibited improved texture and stability.

EXAMPLE 18 Reduction of Ethanol-Soluble and Myristate-Soluble StorageProteins from Triticale, Rye, Barley, Oat, Rice, Sorghum, Corn and Teff

Unless otherwise stated, the materials and methods used in this Exampleare according to those set forth above in the section titled “Reductionof Cereal Proteins, Materials and Methods.”

Triticale, Rye, Barley, Oat and Teff

The reactions were carried out in 30 mM Tris-HCl buffer, pH 7.9. Asindicated, 0.7 μg of NTR and 1 μg of thioredoxin from E. coli or 2 μg ofthioredoxin from yeast, as identified, were added to 70 μL of thisbuffer containing 1 mM NADPH and 25 to 30 μg of extracted storageprotein. The ethanol extracted storage proteins were obtained by using50 ml of 70% ethanol for every 10 gm of flour and extracting for 2 hr.In the case of teff, 200 mg of ground seeds were extracted. Themyristate extracted proteins were obtained by extracting 1 gm of flourwith 8 mg sodium myristate in 5 ml of distilled H₂O for 2 hrs. Thecombination of NADPH, NTR and thioredoxin is known as theNADP/thioredoxin system (NTS). As indicated, glutathione (GSH), 2.5 mM,was added as reductant in either the absence (GSH) or presence of 1.5 mMNADPH and 1.4 μg of spinach leaf glutathione reductase (GR/GSH/NADPH).After incubation for 20 min, 100 nmol of mBBr was added and the reactionwas continued for another 15 min. To stop the reaction and derivatizeexcess mBBr, 10 μL of 10% SDS and 10 μL of 100 mM 2-mercaptoethanol wereadded, and the samples were then applied to the gels. The procedure forSDS-polyacrylamide gel electrophoresis was as described by N. A.Crawford, et al. (1989 Arch. Biochem. Biophys. 271:223-239).

Rice, Sorahum and Corn

The reactions were carried out in 30 mM Tris-HCl buffer, pH 7.9. Whenproteins were reduced by thioredoxin, the following were added to 70 μLof buffer: 1.2 mM NADPH, 10 to 30 μg of seed protein fraction, 0.5 μg E.coli NTR and 1 ug E. coli thioredoxin. For reduction with glutathione,thioredoxin and NTR were replaced with 2.5 mM reduced glutathione and 1μg glutathione reductase (baker's yeast, Sigma Chemical Co.). Forreduction with dithiothreitol, NADPH, thioredoxin, and NTR were omittedand 0.5 mM dithiothreitol was added. In all cases, incubation time was20 min. Then 10 μl of a 10 mM mBBr solution was added and the reactioncontinued for an additional 15 min. To stop the reaction and derivatizeexcess mBBr, 10 μl of 10% SDS and 10 μl of 100 mM 2-mercaptoethanol wereadded and the samples applied to the gels. In each case, to obtain theextracted protein, 1 g ground seeds was extracted with 8 mg of sodiummyristate in 5 ml distilled water. With the exception of the initialredox state determination of the proteins, samples were extracted for 2hr at 22° C. and then centrifuged 20 min at 16,000 rpm prior to theaddition of the mBBr. With the initial redox state determination, themBBr was added under a nitrogen atmosphere along with the myristatefollowed by extraction.

FIGS. 26-30 represent pictures of the gels for the reduction studies ofmyristate-extracted proteins from flour of oat, triticale, rye, barleyand teff. Buffer and ethanol-extracted proteins are also shown for teffin FIG. 30. In all of the studies represented by FIGS. 26-30, the flourwas first extracted with buffer, 50 mM Tris-HCl, pH 7.5 for 20 min. andthen with 70% ethanol for 2 hr. Also shown are pictures of the gels forthe myristate-extracted proteins from corn, sorghum and rice (FIGS. 31and 32). With corn, sorghum and rice, the ground seeds were extractedonly with myristate. Therefore, with corn, sorghum and rice, themyristate extract represents total protein, whereas with oat, triticale,rye, barley and teff, the myristate extract represents only theglutenin-equivalent fractions since these flours had been previouslyextracted with buffer and ethanol. The results, depicted in the gels inFIGS. 26-30, show that the NTS is most effective, as compared to GSH orGSH/GR/NADPH, with myristate-extracted (glutenin-equivalent) proteinsfrom oat, triticale, rye, barley and teff. The NTS is also mosteffective with the total proteins from rice (FIGS. 31 and 32). Reducedglutathione is more effective with the total proteins from corn andsorghum (FIGS. 31 and 32).

Conclusions from FIGS. 31 and 32 (Corn, Sorghum and Rice)

As depicted in FIG. 31 in treatment (1), extraction with myristate inthe presence of mBBr was carried out under a nitrogen atmosphere; intreatment (2), to the myristate extracted proteins mBBr was addedwithout prior reduction of the proteins; in treatment (3), the myristateextracted proteins were reduced by the NADP/thioredoxin system (NTS); intreatment (4) the myristate extracted proteins were reduced by NADPH,glutathione and glutathione reductase. As depicted in FIG. 32, treatment(1) is like treatment (2) in FIG. 31; in treatment (2) the seeds wereextracted with myristate in the presence of mBBr under nitrogen; intreatment (3), seeds were extracted with myristate and reduced by theNTS and then mBBr was added; and in treatment (4) conditions as in (3)except that proteins were reduced by DTT. Treatment (1) in FIG. 31 andtreatment (2) in FIG. 32 show the initial redox state of the proteins inthe grains. For all three cereals, the proteins in the seed are highlyreduced. If extracted in air, the proteins become oxidized especiallythe sorghum and rice. The oxidized proteins can be re-reduced, maximallywith NTS in all cases. With rice, the reduction is relatively specificfor thioredoxin; with corn, glutathione is as effective as thioredoxinand with sorghum glutathione is slightly more effective thanthioredoxin. Dithiothreitol showed varying effectiveness as a reductant.These experiments demonstrate that the storage proteins of these cerealsare less specific than in the case of wheat and suggest that thioredoxinshould be tested both in the presence and absence of glutathione whenattempting to construct a dough network.

FIGS. 33 and 34 represent pictures of the gels resulting from thereduction studies of wheat glutenins and gliadins, respectively, by ayeast NADP/thioredoxin system. The glutenins were obtained by using 50ml of 0.1 M acetic acid for every 10 gm of flour and extracting for 2hr. The gliadins were obtained by using 50 ml of 70% ethanol for every10 gm of flour and extracting for 2 hr. The experiment shows that theyeast system is highly active in reducing the two major groups of wheatstorage proteins.

FIGS. 35-38 represent pictures of gels for the reduction ofethanol-extracted proteins from flour of triticale, rye, oat and barley,respectively. The results show that the NTS is most effective with theethanol-extracted proteins from triticale, rye and oat. Theethanol-extracted barley proteins are reduced in the control andthioredoxin or glutathione has little effect.

EXAMPLE 19 Effect of Thioredoxin-linked Reduction on the Activity andStability of the Kunitz and Bowman-Birk Soybean Trypsin InhibitorProteins Materials and Methods

Plant Materials

Durum wheat (Triticum durum, Desf. cv. Monroe) was a kind gift of Dr. K.Kahn. Wheat germ was obtained from Sigma Chemical Co. (St. Louis, Mo.).

Chemicals and Enzymes

Reagents for sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) were obtained from Bio-Rad Laboratories (Richmond, Calif.),and DTT was from Boehringer Mannheim Biochemicals (Indianapolis, Ind.).L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin(type XIII, T8640), subtilisin (type VIII: bacterial subtilisinCarlsberg, P5380), KTI (T9003), BBTI (T9777), azocasein, and otherchemicals were purchased from Sigma Chemical Co. (St. Louis, Mo.). E.coli thioredoxin and NTR were isolated from cells transformed tooverexpress each protein. The thioredoxin strain containing therecombinant plasmid, pFPI, was kindly provided by Dr. J. -P. Jacquot (deLa Motte-Guery et al., 1991). The NTR strain containing the recombinantplasmid, pPMR21, was kindly provided by Drs. Marjorie Russel and PeterModel (Russel and Model, 1988). The isolation procedures used for theseproteins were as described in those studies with the following changes:cells were broken in a Ribi cell fractionator at 25,000 psi and NTR waspurified as described by Florencio et al. (1988) without the red agarosestep. The E. coli thioredoxin and NTR were, respectively, 100% and 90%pure as determined by SDS-polyacrylamide gel electrophoresis. Wheatthioredoxin h was purified as previously described (Johnson et al.,1987).

Germination of Wheat Seeds

Wheat seeds were sterilized by steeping in 50% (v/v) of Generic Bleachfor 1 h at room temperature, followed by a thorough wash with distilledwater. The sterilized seeds were placed in a plastic Petri dish on twolayers of Whatman filter paper moistened with distilled water containing100 μg/ml of chloramphenicol. Germination was continued at roomtemperature in a dark chamber for up to 5 days.

Preparation of Wheat Proteases

The endosperm (10-15 g fresh weight) isolated from 5-day germinatedwheat seeds by excising the roots and shoots was extracted for 30minutes at 4° C. with 5 volumes of 200 mM sodium acetate, pH 4.6,containing 10 mM β-mercaptoethanol. The homogenate was centrifuged for20 minutes at 48,000 g, 4° C. The pellet was discarded and thesupernatant fluid was fractionated with 30-70% ammonium sulfate. Thisfraction, which represented the protease preparation, was resuspended ina minimum volume of 20 mM sodium acetate, pH 4.6, containing 10 mMβ-mercaptoethanol, and dialyzed against this buffer overnight at 4° C.When assayed with azocasein as substrate, the protease preparation hadan optimal pH of about 4.6 and was stable for at least one week at 4° C.

Reduction and Proteolytic Susceptibility of Trypsin Inhibitors

Unless indicated, the reduction of the trypsin inhibitors (0.4 mg/ml)was carried out in 0.1 ml of 20 mM sodium phosphate buffer, pH 7.9containing 10 mM EDTA at 30° C. for 2 hours. The concentrations ofthioredoxin, NTR, and NADPH were 0.024 mg/ml, 0.02 mg/ml, and 0.25 mM,respectively. With DTT as reductant, EDTA and components of theNADP/thioredoxin system were omitted. Following reduction, aliquots ofthe inhibitor mixture were withdrawn either for determination of trypsininhibitory activity or proteolytic susceptibility. In the subtilisintests, the inhibitor mixture (50 μl) was directly mixed with subtilisinand incubated at room temperature for 1 hour. With the wheat proteasepreparation, the pH of the inhibitor mixture (50 μl) was first adjustedto 4.7 by mixing with 35 μl of 200 mM sodium acetate, pH 4.6; 10 μl ofthe wheat protease preparation was then added and incubation wascontinued for 2 hours at 37° C. To stop digestion with subtilisin, 2 μlof 100 mM phenylmethylsulfonyl fluoride (PMSF) and 10 μl of 10% SDS wereadded to the digestion mixture. With the plant protease preparation,digestion was stopped by adding an equal volume of SDS sample buffer[0.125 M Tris-HCl, pH 6.8, 4% (w/v) SDS, 20% (v/v) glycerol, 10% (v/v)β-mercaptoethanol, and 0.02% (w/v) bromophenol blue]. Proteolyticproducts were analyzed by electrophoresis with 12% or 16% SDSpolyacrylamide slab gels (Laemmli, 1970). The dried slab gels werescanned with a laser densitometer (Pharmacia-LKB UltraScan XL) and thepeak area of the KTI or BBTI protein band was obtained by integrationwith a Pharmacia GelScan XL software program.

Assays

Thioredoxin and NTR were assayed as previously described by Florencio etal. (1988). Trypsin activity was measured in 50 mM Tris-HCl, pH 7.9, byfollowing the increase in absorbance at 253 nm with N-benzoyl-L-arginineethyl ester as substrate (Mundy et al., 1984) or by the release of azodye into the trichloroacetic acid (TCA)-soluble fraction from azocaseinsubstrate (see below). For trypsin inhibition assays, trypsin (5 to 10μg) was preincubated with appropriate amounts of KTI or BBTI for 5minutes at room temperature in 50 mM Tris-HCl, pH 7.9 and proteolyticactivity was then determined. While the two substrates yielded similardata, results are presented with only one substrate.

Wheat protease activity was measured by following the release of azo dyeinto TCA solution from azocasein substrate at pH 4.7. Fifty μl of wheatprotease in a solution of 20 mM sodium acetate, pH 4.6, and 10 mMβ-mercaptoethanol were added to 50 μl of 200 mM sodium acetate, pH 4.6,and 100 μl of 2% azocasein (in 20 mM sodium phosphate, pH 7.0).Following 1-hour incubation at 37° C., 1 ml of 10% TCA was added and themixture was allowed to stand for 10 minutes at room temperature. Aftercentrifugation for 5 minutes in a microfuge (8000 g), 1 ml of thesupernatant solution was withdrawn and mixed with 1 ml of 1 N NaOH. Theabsorbance was read at 440 nm. Protein concentration was determined withBio-Rad reagent using bovine serum albumin as a standard (Bradford,1976).

Results

Trypsin Inhibitory Activity

The 20 kDa Kunitz and 8 kDa Bowman-Birk trypsin inhibitors of soybeancontain 2 and 7 disulfide groups, respectively (Birk, 1976; Wilson,1988). Although their physiological functions have not been established,the two types of inhibitors have been extensively investigated owing totheir wide distribution in legume seeds and their potential to causenutritional disorders, e.g., hypertrophy and associated malfunctions ofthe pancreas. As shown in Tables I and II and described in previousExamples, KTI and BBTI are reduced specifically by the NADP/thioredoxinsystem from either E. coli or plants. The reduced forms of glutathioneand glutaredoxin (a thiol protein capable of replacing thioredoxin incertain animal and bacterial systems, but not known to occur in plants(Holmgren, 1985)) were without effect.

To determine the consequence of reduction by thioredoxin, the trypsininhibitory activity of the oxidized and reduced forms of KTI and BBTIwas compared. As shown in Table XI, preincubation with theNADP/thioredoxin system (NTS) for 2 hours at 30° C. resulted in asubstantial loss of trypsin inhibitory activity (i.e., there was anincrease in trypsin activity relative to the uninhibited control). Morespecifically, the NADP/thioredoxin system effected a 3- and 6-foldincrease in trypsin activity for KTI and BBTI, respectively. Similarresults were obtained with DTT, a nonphysiological substitute forthioredoxin, and with thioredoxin reduced by lipoic acid, a naturallyoccurring dithiol. Extended incubation with DTT alone (overnight at roomtemperature) led to complete or almost complete inactivation of bothinhibitors (data not shown). Unlike DTT, lipoic acid did not reduce(inactivate) KTI and BBTI significantly in the absence of thioredoxin.

TABLE XI Changes in the Ability of Soybean Trypsin Inhibitors to InhibitTrypsin Following Reduction by the NADP/Thioredoxin System, DTT orReduced Lipoic Acid Relative Trypsin Activity* Treatment KTI BBTI Noinhibitor 100 100 Inhibitor Oxidized 17.0 11.5 Reduced by NTS¹ 55.6 70.6Reduced by DTT² 68.6 88.9 Reduced by LA/Trx h³ 40.5 87.8 *The specificactivity of the uninhibited control trypsin was 0.018 ΔA_(253 nm)/μg/minusing N-benzoyl-L-arginine ethyl ester as substrate. ¹Reduction by E.coli NTS (NADP/thioredoxin system) was conducted at 30° C. for 2 hours.²Reduction by DTT (1 mM) was conducted at 30° C. for 1 hour. ³Reductionby lipoic acid (LA, 0.4 mM) and wheat thioredoxin h (Trx h) wasconducted at 30° C. for 1 hour. In the presence of lipoic acid alone(0.4 mM), trypsin activity was 20.0% for KTI and 12.5% for BBTI.

Friedman and colleagues observed that heating soybean flour in thepresence of sulfur reductants (sodium sulfite, N-acetyl-L-cysteine,reduced glutathione, or L-cysteine) inactivated trypsin inhibitors,presumably as a result of the reduction or interchange of disulfidegroups with other proteins in soy flour (Friedman and Gumbmann, 1986;Friedman et al., 1982, 1984). Inactivation of the trypsin inhibitors bythese reductants improved the digestibility and nutritive value offlours in tested rats (Friedman and Gumbman, 1986). Taken together withthese earlier observations, the present findings demonstrate thatdisulfide bonds of both KTI and BBTI targeted by thioredoxin areimportant to maintenance of trypsin inhibitory activity.

Heat Stability

Protease inhibitor proteins are typically stable to inactivationtreatments such as heat. This stability is attributed, at least in part,to the cross-linking of disulfide bonds (Birk, 1976; Ryan, 1981). It isknown that breaking the disulfide bonds by reduction decreases heatstability (Friedman et al., 1982). The question arises as to whetherreduction by thioredoxin yields similar results.

The results as shown in TABLE XII provide a positive answer to thisquestion. When heated at 80° C. for 15 minutes, the thioredoxin-reducedform of KTI completely lost its ability to inhibit trypsin, whereas itsoxidized counterpart retained about half of the original activity (TableXII). Oxidized BBTI was even more stable, retaining the bulk of itstrypsin inhibitory activity after heating at 100° C. for 25 minutes.Nonetheless, as with KTI, the reduced form of BBTI was fully inactivatedby heat (Table XII). These results are consistent with priorobservations (i) that KTI and BBTI show increased sensitivity to heat onreduction; and (ii) that pure BBTI in solution is more heat-stable thanpure KTI in solution. The reverse is true for flour (i.e., KTI is moreheat-stable than BBTI (Friedman et al., 1982 and 1991; and DiPietro andLiener, 1989)).

TABLE XII Heat Stability of the Kunitz and Bowman-Birk TrypsinInhibitors: Oxidized and Following Reduction by the E. coliNADP/thioredoxin System Relative Trypsin Activity* Treatment KTI BBTI Noinhibitor 100 100 Inhibitor, unheated Oxidized 26.6 9.4 Reduced 76.482.4 Inhibitor, heated 15 min at 80° C. Oxidized 52.3 nd¹ Reduced 98.7nd  Inhibitor, heated 25 min at 100° C. Oxidized nd 17.2 Reduced nd 98.4*The specific activity of trypsin was 0.319 ΔA_(440 nm)/mg/min usingazocasein as substrate. The temperatures used for inactivation weredetermined in initial experiments designed to show the heat stability ofthe trypsin inhibitors under out conditions. ¹nd: not determined.

Protease Susceptibility

To test whether the reduced forms of KTI and BBTI show decreasedstability to proteases other than trypsin, both the reduced and oxidizedforms of KTI and BBTI were incubated with a wheat protease preparationor with subtilisin and the proteolytic products were analyzed bySDS-PAGE. The extent of proteolysis was determined by measuring theabundance of intact protein on SDS gels by laser densitometer. Whentested with a protease preparation from 5-day germinated wheat seeds,the oxidized form of the Kunitz inhibitor was almost completelyresistant to digestion whereas the thioredoxin-reduced form wassusceptible to protease. As shown in Table XIII, about 80% of KTI wasdegraded in a reaction that depended on all components of theNADP/thioredoxin system (NTS). BBTI showed the same pattern except thatthe oxidized protein showed greater proteolytic susceptibility relativeto KTI. Similar effects were observed with both inhibitors when theplant protease preparation was replaced by subtilisin (data not shown).The nature of the proteolytic reaction was not investigated, but it isnoted that peptide products were not detected on SDS gels.

TABLE XIII Effect of Thioredoxin-linked Reduction on the Susceptibilityof Kunitz and Bowman-Birk Trypsin Inhibitors to Proteolysis by a PlantProtease Preparation¹ Relative Abundance² Treatment KTI BBTI No protease100 100 Protease No reduction system 97.9 67.2 E. coli NTS³ 22.1 16.0NTS minus thioredoxin 90.2 nd⁴ NTS minus NADPH 97.7 nd  NTS minus NTR97.9 nd  ¹Following reduction by E. coli thioredoxin system at 30° C.for 2 hours, pH was adjusted to 4.7 by addition of 200 mM sodiumacetate, pH 4.6. Wheat protease preparation was then added and incubatedat 37° C. for 2 hours, followed by SDS-PAGE analyses. ²Determined bylaser densitometer. ³NTS: NADP/thioredoxin system. ⁴nd: not determined.

This Example shows that reduction by thioredoxin, or dithiothreitol(DTT), leads to inactivation of both proteins and to an increase intheir heat and protease susceptibility. The results indicate thatthioredoxin-linked reduction of the inhibitor proteins is relevant bothto their industrial processing and to seed germination.

These results confirm the conclusion that disulfide bonds are essentialfor the trypsin inhibitory activity of KTI and BBTI (Birk, 1985;Friedman and Gumbmann, 1986; Friedman et al., 1982, 1984). These studiesalso show that reduction (inactivation) can take place underphysiological conditions (i.e., at low temperature with NADPH-reducedthioredoxin). The ability to inactivate the trypsin inhibitors at lowertemperatures provides a potential method for full inactivation of bothtrypsin inhibitors, thereby improving the quality of soybean productsand saving energy. The need for a method for the complete inactivationof KTI is significant since 20% of its activity is consistently retainedin soy flour under conditions in which BBTI is fully inactivated(Friedman et al., 1991).

The present results also add new information on the proteasesusceptibility of KTI and BBTI. Their increase in proteasesusceptibility following reduction suggests that, if exposed to theprotease inhibitors during seed germination, the NADP/thioredoxin systemcould serve as a mechanism by which the inhibitor proteins are modified(inactivated) and eventually degraded (Baumgartner and Chrispeels, 1976;Chrispeels and Baumgartner, 1978; Orf et al., 1977; Wilson, 1988;Yoshikawa et al., 1979). As stated previously, there is evidence thatthe NADP-thioredoxin system plays a similar role in mobilizing proteinsduring the germination of wheat seeds.

EXAMPLE 20 Reduction of Castor Seed 2S Albumin Protein by Thioredoxin

The results of the following study of sulfhydryl agents to reduce the 2Sprotein from castor seed (Sharief and Li, 1982; Youle and Huang, 1978)shows that thioredoxin actively reduces intramolecular disulfides of the2S large subunit but not the intermolecular disulfides joining the twosubunits.

Materials and Methods

Materials

Seeds of castor (Ricinus communes L. var Hale) were obtained fromBothwell Enterprises, Plainview, Tex.). Biochemicals were obtained fromSigma Chemical Co. (St. Louis, Mo.). E. coli thioredoxin and NTR wereisolated from cells transformed to overexpress each protein. Thethioredoxin strain containing the recombinant plasmid pFPI, was kindlyprovided by Dr. J.-P. Jacquot (de La Mott-Guery et al. 1991). The straincontaining the recombinant plasmid, pPMR21, was kindly provided by Drs.Marjorie Russel and Peter Model (Russel and Model, 1988). Thioredoxinand NTR were purified by the respective procedures of de La Mott-Gueryet al. (1991) and Florencio et al. (1988). Reagents forSDS-polyacrylamide gel electrophoresis were purchased from Bio-RadLaboratories (Richmond, Calif.). Monobromobimane (mBBr) or Thiolite wasobtained from Calbiochem (San Diego, Calif.). Other chemicals wereobtained from commercial sources and were of the highest qualityavailable. NADP-malate dehydrogenase and fructose-1,6-bisphosphatasewere purified from leaves of corn (Jacquot et al. 1981) and spinach(Nishizawa et al. 1982), respectively. Thioredoxin h was isolated fromwheat seeds by following the procedure devised for the spinach protein(Florencio et al. 1988). Glutathione reductase was prepared from spinachleaves (Florencio et al. 1988).

Isolation of Protein Bodies

Protein bodies were isolated by a nonaqueous method (Yatsu and Jacks,1968). Shelled dry castor seeds, 15 g, were blended with 40 ml ofglycerol for 30 sec in a Waring blender. The mixture was filteredthrough four layers of nylon cloth. The crude extract was centrifuged at272×g for 5 min in a Beckman J2-21M centrifuge using a JS-20 rotor.After centrifugation, the supernatant fraction was collected andcentrifuged 20 min at 41,400×g. The pellet, containing the proteinbodies, was resuspended in 10 ml glycerol and centrifuged as before(41,400×g for 20 min) collecting the pellet. This washing step wasrepeated twice. The soluble (“matrix”) fraction was obtained byextracting the pellet with 3 ml of 100 mM Tris-HCl buffer (pH 8.5). Theremaining insoluble (“crystalloid”) fraction, collected bycentrifugation as before, was extracted with 3 ml of 6M urea in 100 mMTris-HCl buffer (pH 8.5).

2S Protein Purification Procedure

The 2S protein was prepared by a modification of the method of Tully andBeevers (1976). The matrix protein fraction was applied to aDEAE-cellulose (DE-52) column equilibrated with 5 mM Tris-HCl buffer, pH8.5 (Buffer A) and eluted with a 0 to 300 mM NaCl gradient in buffer A.Fractions containing the 2S protein were pooled and concentrated byfreeze drying. The concentrated fraction was applied to a Pharmacia FPLCSuperose-12 (HR 10/30) column equilibrated with buffer A containing 150MM NaCl. The fraction containing 2S protein from the Superose-12 columnwas applied to an FPLC Mono Q HR 5/5 column equilibrated with buffer A.The column was eluted sequentially with 3 ml of buffer A, 20 ml of alinear gradient of 0 to 300 mM NaCl in buffer A and finally with bufferA containing 1 M NaCl. The 2S protein purified by this method was freeof contaminants in SDS polyacrylamide gels stained with Coomassie blue(Kobrehel et al., 1991).

Analytical Methods

Reduction of proteins was monitored by the monobromobimane (mBBr)/SDSpolyacrylamide gel electrophoresis procedure of Crawford et al. (1989).Labeled proteins were quantified as described previously in the“Reduction of Cereal Proteins, Materials and Methods” section. Proteinwas determined by the method of Bradford (1976).

Enzyme Assays/Reduction Experiments

The Wada et al., 1981 protocol was used for assaying NADP-malatedehydrogenase and fructose 1,6 bisphosphatase in the presence ofthioredoxin and 2S protein. Assays were conducted under conditions inwhich the amount of added thioredoxin was sufficient to reduce thecastor 2S protein but insufficient to activate the target enzymeappreciably. All assays were at 25° C. Unless otherwise indicated, thethioredoxin and NTR used were from E. coli. The 2S protein was monitoredduring purification by mBBr/SDS-polyacrylamide gel electrophoresisfollowing its reduction by dithiothreitol and E. coli thioredoxin(Crawford et al., 1989; Kobrehel et al., 1991).

FIG. 39 represents the reduction of the matrix and crystalloid proteinsfrom castor seed as determined by mBBr/SDS-polyacrylamide gelelectrophoresis procedure. 1 and 7, Control: no addition; 2 and 8,GSH/GR/NADPH: reduced glutathione, glutathione reductase (from spinachleaves) and NADPH; 3 and 9, NGS: NADPH, reduced glutathione, glutathionereductase (from spinach leaves) and glutaredoxin from E. coli; 4 and 10,NTS: NADPH, NTR, and thioredoxin (both proteins from E. coli); 5 and 11,NADPH; 6 and 12, NADPH and E. coli NTR. Reactions were carried out in100 mM Tris-HCl buffer, pH 7.8. As indicated, 0.7 μg NTR and 1 μgthioredoxin were added to 70 μl of this buffer containing 1 mM NADPH andtarget protein: 8 μg matrix protein for treatments 1-6 and 10 μgcrystalloid protein for treatments 7-12. Assays with glutathione wereperformed similarly, but at a final concentration of 2 mM, 1.4 μgglutathione reductase, 1 μg glutaredoxin, and 1.5 mM NADPH. Reactiontime was 20 min.

FIG. 40 represents the specificity of thioredoxin for reducing thedisulfide bonds of castor seed 2S protein. (1) Control (no addition);(2) Control+NTS (same conditions as in FIG. 39); (3) Control (heated 3min at 100° C.), (4) Control+2 mM DTT (heated 3 min at 100° C.). Thesamples containing 5 μg 2S protein and the indicated additions wereincubated for 20 min in 30 mM Tris-HCl (pH 7.8). mBBr, 80 nmol, was thenadded and the reaction continued for another 15 min prior to analysis bythe mBBr/SDS polyacrylamide gel electrophoresis procedure.

Results

The castor storage proteins, which are retained within a protein bodyduring seed maturation, can be separated into two fractions on the basisof their solubility. The more soluble proteins are housed in the proteinbody outer section (“matrix”) and the less soluble in the inner(“crystalloid”). In the current study, the matrix and crystalloidcomponents were isolated to determine their ability to undergo reductionby cellular thiols, viz., glutathione, glutaredoxin and thioredoxin.Glutaredoxin, a 12 kDa protein with a catalytically active thiol group,can replace thioredoxin in certain enzymatic reactions of bacteria andanimals (Holmgren et al. 1985) but is not known to occur in plants.

FIG. 39 shows that, while a number of storage proteins of castor seedwere reduced by the thiols tested, only a low molecular weight protein,corresponding to the large subunit of the 2S protein of the matrix,showed strict specificity for thioredoxin. Certain higher molecularweight proteins of the crystalloid fraction underwent reduction, but inthose cases there was little difference between glutaredoxin andthioredoxin (FIG. 39). The castor seed 2S large subunit thus appeared toresemble cystine-containing proteins previously discussed in undergoingthioredoxin-specific reduction. These experiments were designed toconfirm this specificity and to elucidate certain properties of thereduced protein. As expected, owing to lack of disulfide groups, the 2Ssmall subunit showed essentially no reaction with mBBr with any of thereductants tested.

When its fluorescent band was monitored by laser densitometry, thereduction of the castor seed 2S large subunit was found to depend on allcomponents of the NADP/thioredoxin system (NADPH, NTR and thioredoxin)(Table XIV). As for other thioredoxin-linked proteins (includingchloroplast enzymes), the thioredoxin active in reduction of the 2Slarge subunit could be reduced either chemically with dithiothreitol(DTT) or enzymatically with NADPH and NTR. The extent of reduction bythe NADP thioredoxin system, DTT alone, and DTT+thioredoxin was 84%, 67%and 90%, respectively, after 20 min at 25° C. Similar, though generallyextensive reduction was observed with the disulfide proteins discussedabove (Johnson et al. 1987). As with the other seed proteins, nativewheat thioredoxin h and E. coli thioredoxins could be usedinterchangeably in the reduction of the 2S protein by DTT (data notshown).

TABLE XIV

Extent of reduction of the castor castor seed 2S protein by differentsulfhydryl reductants. Reaction conditions as in FIG. 39. A reduction of100% corresponds to that obtained when the 2S protein was heated for 3min in the presence of 2% SDS and 2.5% β-mercaptoethanol. NTS: NADPH,NTR, and thioredoxin (both proteins from E. coli); GSH/GR/NADPH: reducedglutathione, glutathione reductase (from spinach leaves) and NADPH; NGS:NADPH, reduced glutathione, glutathione reductase (from spinach leaves)and glutaredoxin (from E. coli).

Treatment Relative Reduction, % Control 0 NADP/thioredoxin system,complete 84 NADP/thioredoxin system, minus thioredoxin 0NADP/thioredoxin system, minus NADPH 0 NADP/thioredoxin system, minusNTR 0 Reduced glutathione 0 NADP/glutaredoxin system, complete 0 DTT 67DTT + Thioredoxin 90

The capability of thioredoxin to reduce the castor seed 2S protein wasalso evident in enzyme activation assays. Here, the protein targeted bythioredoxin (in this case 2S) is used to activate a thioredoxin-linkedenzyme of chloroplasts, NADP-malate dehydrogenase or fructose1,6-bisphosphatase. As with most of the proteins examined so far, the 2Sprotein more effectively activated NADP-malate dehydrogenase and showedlittle activity with the fructose bisphosphatase (2.6 vs. 0.0nmoles/min/mg protein).

The castor seed 2S protein contains inter-as well as intramoleculardisulfides. The 2S protein thus provides an opportunity to determine thespecificity of thioredoxin for these two types of bonds. To this end,the castor seed 2S protein was reduced (i) enzymically with theNADP/thioredoxin system at room temperature, and (ii) chemically withDTT at 100° C. Following reaction with mBBr the reduced proteins wereanalyzed by SDS-polyacrylamide gel electrophoresis carried out withoutadditional sulfhydryl agent. The results (FIG. 40) indicate that whilethioredoxin actively reduced intramolecular disulfides, it was much lesseffective with intermolecular disulfides.

The present results extend the role of thioredoxin to the reduction ofthe 2S protein of castor seed, an oil producing plant. Thioredoxinspecifically reduced the intramolecular disulfides of the large subunitof the 2S protein and showed little activity for the intermoleculardisulfides joining the large and small subunits. Based on the resultswith the trypsin inhibitors of soybean, it is clear that reduction ofintramolecular disulfides by thioredoxin markedly increases thesusceptibility of disulfide proteins to proteolysis (Jiao et al. 1992a).It, however, remains to be seen whether reduction of the 2S proteintakes place prior to its proteolytic degradation (Youle and Huang, 1978)as appears to be the case for the major storage proteins of wheat. Arelated question raised by this work is whether the 2S protein ofcastor, as well as other oil producing plants such as brazil nut(Altenbach et al., 1987; Ampe et al., 1986), has a function in additionto that of a storage protein.

EXAMPLE 21 Thioredoxin-Dependent Deinhibition of Pullulanase of Cerealsby Inactivation of a Specific Inhibitor Protein

Assay of Pullulanase

1. Standard Curve of Maltotriose

A series of concentrations of maltotriose (0 to 2 mg) in 0.1 to 0.2 mlwater or buffer were made in microfuge tubes. To this was added 0.2 mlof dinitrosalicylic acid (DA) reagent (mix 1 g of DA, 30 g of sodiumpotassium tartrate, and 20 ml of 2N NaOH with water to final volume of100 ml). The reagents were dissolved in a warm water bath. The mixturewas heated at 100° C. for 5 min and cooled down in a water bath (roomtemperature). Each sample was transferred to a glass tube that contained2 ml of water. Read A₄₉₃ vs water. ΔA₄₉₃ [A₄₉₃ of sample containingmaltotriose was subtracted from A₄₉₃ of the blank (no maltotriose)] wasplotted against maltotriose concentrations.

2. Pullulanase Activity Assay

Pullulanase activity is measured as the release of reducing sugar fromthe substrate pullulan. Typically 10-100 μl of pullulanase sample (in 20mM Tris-HCl, pH 7.5, or in 5-20 acetate-NA, pH 4.6) was mixed with25-100 μl of 200 mM Acetate-NA, pH 5.5 (this buffer serves to bringfinal pH of the assay to 5.5) and 10-20 μl of 2% (w/v) pullulan. Themixture was incubated at 37° C. for 30 to 120 min, depending on theactivity of pullulanase. The reaction was stopped by adding 200 μl of DAreagent. Reducing sugar was then determined as above.

Note

1. When a crude extract of pullulanase obtained by the dialysis of crudeextracts or pullulanase obtained from a dialyzed 30-60% ammonium sulfatefraction is used as a pullulanase source, it must be thoroughly dialysedbefore assay because there are reduced sugars in the crude extract. Inother words the background of crude pullulanase samples from dialysedcrude extracts or a dialysed 30-60% ammonium sulfate fraction is veryhigh. In this case, the blank is made as follows: 200 μl of DA reagentare added first, followed by the addition of enzyme sample, pullulan andbuffer.

2. When final concentrations of DTT (or β-mercaptoethanol (MET) or GSH)are higher than 2 mM in the assay mixture, the OD₄₉₃ values will begreater than those of the minus-DTT (MET, GSH) samples. DTT (MET, GSH)should be added to the blank, samples without DTT during assay at theend of the reaction. Care should be taken to make sure the finalconcentration of DTT in the assay mixture is below 2 mM.

Purification of Pullulanase Inhibitor Extraction and Ammonium SulfateFractionation

200 g of barley malt was ground to fine powder with an electric coffeegrinder and extracted with 600 ml of 5% (w/v) NaCl for 3 h at 30° C.Following centrifugation at 30,000 g and at 4° C. for 25 min, thesupernatant was fractionated by precipitation with solid ammoniumsulfate. Proteins precipitated between 30% and 60% saturated ammoniumsulfate were dissolved in a minimum volume of 20 mM Tris HCl, pH 7.5,and dialyzed against this buffer at 4° C. overnight.

DE52 Chromatography

The dialyzed sample was centrifuged to remove insoluble materials andthe supernatant adjusted to pH 4.6 with 2N formic acid. After pelletingthe acid-denatured protein, the supernatant was readjusted to pH 7.5with NH₄OH and loaded onto a DE52 column (2.5×26 cm) equilibrated with20 mM Tris-HCl, pH 7.5. Following wash with 80 ml of the above buffer,the column was eluted with a linear 0-500 mM Tris-HCl, pH 7.5. Fractionsof 6.7 ml were collected. Pullulanase was eluted at about 325 mM NaCland its inhibitor came off at about 100 mM NaCl. Pullulanase was furtherpurified through CM32 (20 mM sodium acetate, pH 4.6) and Sephacryl-200HR (30 mM Tris-HCl, pH 7.5, containing 200 mM NaCl and 1 mM EDTA)chromatography. Pullulanase inhibitor protein was purified as describedbelow.

CM32 Chromatography

The pullulanase inhibitor sample (about 90 ml) from the DE52 step wasplaced in a 150-ml flask and incubated at 70° C. water-bath for 20 min.Following centrifugation, the clarified sample was then adjusted to pH4.6 with 2N formic acid and dialyzed against 20 mM sodium acetate, pH4.6. The precipitate formed during dialysis was removed bycentrifugation and the supernatant was chromatographed on a CM32 column(2.5×6 cm) equilibrated with 20 mM sodium acetate, pH 4.6. Proteins wereeluted with a linear 0-0.4 M NaCl in 200 ml of 20 mM sodium acetate, pH4.6. Fractions (5.0 ml/fraction) containing pullulanase inhibitoryactivity were pooled, dialyzed, and rechromatographed on a CM32 column(2.5×6 cm) with a linear 0.2-1 M NaCl gradient in 200 ml of 20 mM sodiumacetate, pH 4.0.

Sephadex G-75 Filtration

Pullulanase inhibitor fractions from the second CM32 chromatography stepwere concentrated in a dialysis bag against solid sucrose and thenseparated by a Sephadex G-75 column (2.5×85 cm) equilibrated with 30 mMTris-HCl, pH 7.5, containing 200 mM Na Cl and 1 mM EDTA. Fractions (3.6ml/fraction) showing pullulanase inhibitory activity were pooled,concentrated by dialysis against solid sucrose, and then dialysedagainst 10 mM Tris-HCl, pH 7.5.

Identification and Purification of Pullulanase Inhibitor

During germination, starch is converted to glucose by α-, β-amylases,and pullulanase (also called debranching enzyme, R-enzyme). Whileextensive studies have been conducted for the regulation of amylases,little is known about the regulation of pullulanase in seeds. Yamada(Yamada, J. (1981) Carbohydrate Research 90:153-157) reported thatincubation of cereal flours with reductants (e.g., DTT) or proteases(e.g., trypsin) led to an activation of pullulanase activity, suggestingthat reduction or proteolysis might be a mechanism by which pullulanaseis activated during germination. Like in rice flour, pullulanaseextracts from germinated wheat seeds or from barley malt showed loweractivity, and were activated 3 to 5-fold by preincubation with DTT for20 to 30 min. However, following purification of the crude extract (adialysate of 30-60% ammonium sulfate fraction) by anion or cationexchange chromatography, the total pullulanase activity increased 2 to3-fold over that of the sample applied to the column when assayedwithout preincubation with DTT, and DTT has no or little effect onpullulanase. One possibility was that pullulanase might be activated byproteolysis during the process of purification, since germinated wheatseeds or barley malt show high protease activity. If this was the case,addition of protease inhibitor cocktail would prevent pullulanaseactivation during purification. In contrast to this point, manyexperiments with protease inhibitors failed to prove this. Anotherpossibility was that there is an inhibitor that is precipitated byammonium sulfate and inhibits pullulanase. The role of DTT is to reduceand thus inactivate this protein inhibitor, leading to activation ofpullulanase. Along this line, the 30-60% ammonium sulfate fraction frombarley malt was applied to a DE52 column (2.5×26 cm) equilibrated with20 mM Tris-Cl, pH 7.5 (FIG. 41). Following elution with a linear saltgradient, “deinhibited” (“activated”) pullulanase was identified as aprotein peak coming off at about 325 mM NaCl (from fraction numbers 44to 60). Assay of pullulanase activity in the preincubation mixtureconsisting of 50 μl of the peak pullulanase activity fraction (fractionnumber 45) with 50 μl of other protein fractions indicated that aprotein peak that showed pullulanase inhibitory activity was eluted fromthe DE52 column by about 100 mM NaCl between fraction numbers 8 to 25(FIG. 41).

The pullulanase inhibitor sample was further purified by two consecutivecation exchange chromatography steps with CM32 at pH 4.6 (FIG. 42) and4.0 (FIG. 43) and filtration with Sephadex G-75 (FIG. 44).

Properties of Pullulanase Inhibitor

Preliminary experiments showed that pullulanase inhibitor protein isresistant to treatment of 70° C. for 10 min and pH 4.0. Based on theprofile of Sephadex G-75 gel filtration and SDS-PAGE, pullulanaseinhibitor has a molecular weight between 8 to 15 kDa±2 kDa. The studyfurther showed that the protein contains thioredoxin-reducible (S—S)bonds.

These studies, as shown in Table XV, found that the ubiquitous dithiolprotein, thioredoxin, serves as a specific reductant for a previouslyunknown disulfide-containing protein that inhibits pullulanase of barleyand wheat endosperm.

TABLE XV Activity change in Pullulanase Inhibitor Protein FollowingReduction by NADP/Thioredoxin System Relative Pullulanase TreatmentActivity No inhibitor 100 Inhibitor Oxidized 30.1 Reduced by DTT 46.1Reduced by E. coli Trx/DTT 95.1 Reduced by E. coli NTS 40.4 Reduced byGSH/NADPH/GR 33.6

Reduction of the inhibitor protein eliminated its ability to inhibitpullulanase, thereby rendering the pullulanase enzyme active. Thesestudies as shown in Table XV illustrate that it is possible to renderthe pullulanase enzyme active with a physiological system consisting ofNADPH, NADP-thioredoxin reductase (NTR) and thioredoxin (theNADP/thioredoxin system) as well as with thioredoxin (Trx) anddithiothreitol. These findings also elucidate how reductive activationof pullulanase takes place (i.e., that a specific (previously unknown)inhibitor is reduced and thereby inactivated, so that the enzyme becomesactive). The thioredoxin active in this reaction can be obtained fromseveral sources such as E. coli or seed endosperm (thioredoxin h). Therole of thioredoxin in reductively inactivating the inhibitor protein(I) and deinhibiting the pullulanase enzyme (E) is given in Equations 1and 2.

In summary, the crude endosperm extracts were fractionated by columnchromatography procedures. These steps served to separate the proteininhibitor from the pullulanase enzyme. The inhibitor protein was thenhighly purified by several steps. By use of the mBBr/SDS-PAGE procedure,it was determined that disulfide group(s) of the new protein arespecifically reduced by thioredoxin and that the reduced protein losesits ability to inhibit pullulanase. Like certain other disulfideproteins of seeds (e.g., the Kunitz and Bowman-Birk trypsin inhibitorsof soybean), the pullulanase inhibitor protein showed the capability toactivate chloroplast NADP-malate dehydrogenase. In these experiments,dithiothreitol was used to reduce thioredoxin, which in turn reducedinhibitor and thereby activated the dehydrogenase enzyme.

EXAMPLE 22 Engineering of Yeast Cells to Overexpress Thioredoxin andNADP-Thioredoxin Reductase

The two Saccharomyces cerevisiae thioredoxin genes (Muller, E. G. D.(1991), J. Biol. Chem. 266:9194-9202), TRX1 and TRX2, are cloned in highcopy number episomal vectors, an example of which is YEp24, under thecontrol of strong constitutive promoter elements, examples of which arethe glycolytic promoters for the glyceraldehyde-3-P dehydrogenase,enolase, or phosphoglycerate kinase genes. Recombinant constructs areassessed for the overexpression of thioredoxin by quantitative Westernblotting methods using an antithioredoxin rabbit antiserum (Muller, E.G. D., et al. (1989), J. Biol. Chem. 264:4008-4014), to select theoptimal combination of thioredoxin genes and promoter elements. Thecells with the optimal thioredoxin overexpression system are used as asource of thioredoxin for dough improvement.

The NADP-thioredoxin reductase gene is cloned by preparing anoligonucleotide probe deduced from its amino terminal sequence. Theenzyme is prepared from yeast cells by following a modification of theprocedure devised for spinach leaves (Florencio, F. J., et al. (1988),Arch. Biochem. Biophys. 266:496-507). The amino terminus of the purereductase enzyme is determined by microsequencing by automated Edmandegradation with an Applied Biosystems gas-phase protein sequencer. Onthe basis of this sequence, and relying on codon usage in yeast, a20-base 24-bold degenerate DNA probe is prepared. The probe ishybridized to isolated yeast DNA cleaved with EcoRI and PstI by Southernblot analysis. The most actively region is extracted from the agarosegels and introduced into a pUC19 plasmid vector (Szekeres, M., et al.(1991), J. Bacteriol. 173:1821-1823). Following transformation,plasmid-containing E. coli colonies are screened by colony hybridizationusing the labeled oligonucleotide probe (Vogeli, G., et al. (1987),Methods Enzymol. 152:407-415). The clone is identified as carrying thegene for NADP-thioredoxin reductase by sequencing the DNA as given inSzekeres, et al. above. Once identified, the NADP-thioredoxin reductasegene is overexpressed in yeast as described above for the TRX1 and TRX2yeast thioredoxin genes. The yeast cells which overexpressNADP-thioredoxin reductase are used as a source of reductase to improvedough quality.

EXAMPLE 23 Improvement in Dough Quality Using Genetically EngineeredYeast Cells

Saccharomyces cerevisiae cells engineered to overexpress the two yeastthioredoxins and the yeast NADP-thioredoxin reductase as set forth inExample 23 are lysed by an established procedure such as sonication andthen freeze dried. The dried cells from the cultures overexpressingthioredoxin and NADP-thioredoxin reductase are combined and then used tosupplement flour to improve its dough quality. Two-tenths gram of thecombined lysed dried cells are added together with about 300 to about500 nanomoles NADPH to 1 M Tris-HCl buffer, pH 7.9, to give 5.25 ml of30 mM Tris-HCl. The reaction is carried out in a microfarinograph at 30°C. as described in Example 14. An improvement in dough quality isobserved which is similar to the improvement shown in Example 14.

EXAMPLE 24 Improvement of Gluten

The positive effects of the NADP/thioredoxin system on dough qualitypresents the option of applying this system to flour in the preparationof gluten. The purpose is to alter the yield and the properties ofgluten, thereby enhancing its technological value: (1) by obtainingstronger glutens (increased elasticity, improved extensibility); (2) byincreasing gluten yield by capturing soluble proteins, reduced by theNADP-thioredoxin system, in the protein network, thereby preventing themfrom being washed out during the production of gluten. In this procedure(using 10 g flour), 0.2 μg E. coli thioredoxin, 0.1 μg E. coliNADP-thioredoxin reductase and 300 to 500 nanomoles NADPH are addedtogether with 1 M Tris-HCl, pH 7.9, buffer to give 5.25 ml of 30 mMTris-HCl. The gluten is made at room temperature according to the commonlixiviation method. The yield of the gluten is determined by weight andthe strength of the gluten is determined by the classical manual stretchmethod. The gluten product which are obtained by this treatment with theNADP/thioredoxin system is used as an additive in flour or other grain.

EXAMPLE 25 Method of Producing Dough from a Non-wheat or Rye Flour

For this test (using 10 gm of milled flour from corn, rice or sorghum),0.2 μg E. coli thioredoxin, 0.1 μg E. coli NADP-thioredoxin reductaseand 500 nanomoles NADPH are added together with 1 M Tris-HCl, pH 7.9,buffer to give 5.25 ml of 30 mM Tris-HCl. The reaction is carried out bymixing the 10 gm of milled flour with the enzyme system in amicro-farinograph at 30° C. The farinograph measurements show wheat-likedough characteristics by the added NADP-thioredoxin system. In thecontrols without the enzyme system, no microfarinograph reading ispossible because the mixture fails to form a dough. The dough which isformed is persistent and its consistency is maintained throughout therun. The end product is similar to the network formed in dough derivedfrom wheat.

Reduction of Animal Toxins

The invention provides a method for chemically reducing toxicity causingproteins contained in bee, scorpion and snake venoms and therebyaltering the biological activity of the venoms well as reducing thetoxicity of animal toxins specifically snake neurotoxins by means ofthiol redox (SH) agents namely a reduced thioredoxin, reduced lipoicacid in the presence of a thioredoxin or DTT. The reduction of thethioredoxin occurs preferably via the NADP-thioredoxin system (NTS). Asstated previously, the NTS comprises NADPH, NADP-thioredoxin reductase(NTR) and a thioredoxin.

The term “thiol Redox agent” has been used sometimes in the literatureto denote both an agent in the nonreduced state and also in the reducedor sulfhydryl (SH) state. As defined herein the term “thiol redox (SH)agent” means a reduced thiol redox protein or synthetically preparedagent such as DTT.

The reduction of the neurotoxin may take place in a medium that isliquid such as blood, lymph or a buffer, etc. or in a medium that issolid such as cells or other living tissue. As used herein the term“liquid” by itself does not refer to a biological fluid present in anindividual.

Presumably the proficiency of the thiol redox (SH) agents to inactivatethe venom in vitro and to detoxify the venom in individuals depends uponthe ability of the agents of the invention to reduce the intramoleculardisulfide bonds in these toxicity causing venom components.

All snake neurotoxins, both presynaptic and postsynaptic can be reducedand at least partially inactivated in vitro by the thiol redox (SH)agents of the invention. Snake neurotoxins inactivated in vitroaccording to the invention are useful as antigens in the preparation ofantivenoms. The neurotoxins are inactivated preferably by incubationwith a thiol redox (SH) agent in an appropriate buffer. The preferredbuffer is Tris-HCl buffer but other buffers such as phosphate buffer maybe used. The preferred thiol redox (SH) agent is a reduced thioredoxin.

Effective amounts for inactivating snake neurotoxins range from about0.1 μg to 5.0 μg, preferrably about 0.5 μg to 1.0 μg, of a reducedthioredoxin; from about 1 nanomole to 20 nanomoles, preferably from 5nanomoles to 15 nanomoles, of reduced lipoic acid in the presence ofabout 1.0 μg of a thioredoxin and from about 10 nanomoles to 200nanomoles, preferably 50 nanomoles to 100 nanomoles, of reduced DTT(preferably in the presence of about 1.0 μg of a thioredoxin) for every10 μg of snake neurotoxin in a volume of 100 μl.

The effective amounts for inactivating a snake neurotoxin using thecomponents in the NADP-thioredoxin system range from about 0.1 μg to 5.0μg, preferably about 0.5 μg to 1.0 μg, of thioredoxin; from about 0.1 μgto 2.0 μg, preferrably from 0.2 μg to 1.0 μg, of NTR and from about 0.05micromoles to 0.5 micromoles, preferably about 0.1 micromoles to 0.25micromoles, of NADPH for every 10 μg of snake neurotoxin in a volume of100 μl.

Upon inactivation the buffer containing the inactivated neurotoxin andthiol redox (SH) agent, etc. may be injected into an animal such as ahorse to produce an antivenom or prior to injection it may be furthertreated with heat or formaldehyde.

The thiol redox (SH) agents of the invention may also be used to treatindividuals who are suffering the effects of neurotoxicity caused by avenomous snake bite. The preferred method of administering the reducedthiol redox (SH) agent to the individual is by multiple subcutaneousinjections around the snake bite.

Of course the correct amount of a thiol redox (SH) agent used todetoxify a neurotoxin in an individual will depend upon the amount oftoxin the individual actually received from the bite. However, effectiveamounts for detoxifying or reducing the toxicity of snake neurotoxins inmice usually range from about 0.01 μg to 0.3 μg, preferably about 0.02μg to 0.05 μg, of a reduced thioredoxin; from about 0.1 nanomole to 3.0nanomoles, preferably from 0.2 nanomole to 1.0 nanomole, of reducedlipoic acid in the presence of about 0.05 μg of a thioredoxin; fromabout 1.0 nanomole to 30 nanomoles, preferably from 2.0 nanomoles to 5.0nanomoles, of DTT, preferably in the presence of 0.05 μg of athioredoxin, for every gm of mouse body weight.

The effective amounts for detoxifying a snake neurotoxin in a mouseusing the components of the NADP-thioredoxin system range from about0.01 μg to 0.3 μg, preferably about 0.02 μg to 0.05 μg of a thioredoxin;from about 0.005 μg to 0.12 μg, preferably from 0.01 μg to 0.025 μg, ofNTR and from about 5 nanomoles to 30 nanomoles, preferably 10 nanomolesto 15 nanomoles, NADPH for every gm of mouse body weight.

The preferred method of administering the NTS to an individual is alsoby multiple subcutaneous injections. The preferred thiol redox agent forhuman use is human thioredoxin administered via the NADP-thioredoxinsystem or with lipoic acid or DTT.

A partial list of the venomous snakes which produce the neurotoxinswhich can be inactivated or detoxified by the methods of this inventionappears on pages 529-555 of Chippaur, J.-P., et al. (1991) ReptileVenoms and Toxins, A. T. Tu, ed., Marcel Dekker, Inc., which is hereinincorporated by reference.

Other features and advantages of the invention with respect toinactivating and detoxifying venoms can be ascertained from thefollowing examples.

EXAMPLE 26 Reduction Studies of Bee, Scorpion and Snake Venoms andLabeling with mBBr

Reactions were carried out with 50 μg venom (final volume of 100 μl) in30 mM Tris-Cl buffer pH 7.9 containing the following proteaseinhibitors: phenylmethylsulfonyl fluoride (PMSF), leupeptin andpepstatin (final concentrations used in the assay respectively: 100 μM,1 μM and 1 μM). With NADPH as a reductant, the mixture also contained 4μg thioredoxin, 3.5 μg NTR (both from E. coli) and 12.5 mM NADPH. Whenthioredoxin (4 μg, E. coli or human) was reduced by DTT, NADPH and NTRwere omitted and DTT was added to 0.5 mM. Assays with GSH were performedsimilarly but at a final concentration of 5 mM and in the presence of1.5 μg glutathione reductase and 12.5 mM NADPH. The mixture wasincubated for 20 min at room temperature, mBBr was then added to 1.5 mMand the reaction was continued for 15 min at room temperature. Thereaction was stopped and excess mBBr derivatized by adding 10 μl of 100mM β-mercaptoethanol, 5 μl of 20% SDS and 10 μl of 50% glycerol. Sampleswere then analyzed by SDS-polyacrylamide gel electrophoresis aspreviously described.

The same experiment with the NADP-thioredoxin system was performedwithout adding protease inhibitors.

The extent of the reduction of the bee, scorpion and snake venoms bydifferent reductants described above is shown in FIGS. 45, 46 and 47,respectively. FIGS. 45, 46 and 47 represent the results of the reductionstudies of different venoms (SDS-Polyacrylamide gel/mBBr procedure).After 20 min incubation at room temperature with different reductantsand in the presence of protease inhibitors, the samples were derivatizedwith mBBr and separated by electrophoresis and fluorescence wasdetermined. (FIG. 45: Bee venom from Apis mellifera; FIG. 46: scorpionvenom from Androctonus australis, and FIG. 47: snake venom from Bungarusmulticinctus). It may be seen that in all these cases thioredoxin (E.coli or human) specifically reduced components of the venoms. FIG. 48shows that thioredoxin reduces venom components in a similar way whenthe reaction was performed in the absence of protease inhibitors.

FIG. 48 represents the results of the reduction of bee, scorpion andsnake venoms by the NADP-Thioredoxin system with and without proteaseinhibitors (SDS-Polyacrylamide gel mBBr procedure). After 20 minincubation at room temperature with NTS in the presence or absence ofany protease inhibitors, the samples were derivatized with mBBr,separated by electrophoresis, and fluorescence was determined as inFIGS. 45-47.

Materials

Venoms: Bee venom from Apis mellifera, scorpion venom from Androctonusaustralis, and snake venom from Bungarus multicinctus were purchasedfrom Sigma chemical Co. (St. Louis, Mo.).

Protease Inhibitors: Phenylmethylsulfonyl fluoride (PMSF), Leupeptin andPepstatin were purchased from Sigma Chemical Co. (St. Louis, Mo.).

Venom Detoxification

Detoxification of bee, scorpion and snake venoms is determined bysubcutaneous injection into mice. Assays are done in triplicate. Priorto injection, the venom is diluted in phosphate-saline buffer (0.15 MNaCl in 10 mM Na₂HPO₄/NaH₂PO₄ pH 7.2) at concentrations ranging up totwice the LD₅₀ (per g mouse): bee venom from Apis mellifera, 2.8 μg;scorpion venom from Androctonus australis, 0.32 μg; and snake venom fromBungarus multicinctus, 0.16 μg. At 5, 10, 30, 60 minutes and 4, 12 and24 hr after injection, separate groups of challenged mice are injected(1) intravenously and (2) subcutaneously (multiple local injectionsaround the initial injection site). The thioredoxin is reduced with: (1)E. coli NADP-thioredoxin system, using 0.08 μg thioredoxin, 0.07 μg NTRand 25 nmoles NADPH; (2) Thioredoxin reduced by DTT or reduced lipoicacid (0.08 μg E. coli or human thioredoxin added to 1 nmoledithiothreitol or 1 nmole of reduced lipoic acid). Concentrations areper μg venom injected into the animal; all solutions are prepared inphosphate-saline buffer.

The effect of thioredoxin on detoxification is determined by (1)comparing the LD₅₀ with the control group without thioredoxin and (2)following the extent of the local reaction, as evidenced by necrosis,swelling and general discomfort to the animal.

Reduction Studies for Reducing Snake Neurotoxins—Materials and Methods

Toxins

Porcine pancreas phospholipase A₂, erabutoxin b and β-bungarotoxin werepurchased from Sigma Chemical Co. (St. Louis, Mo.). As the phospholipaseA₂ was provided in 3.2 M (NH₄)₂SO₄ solution pH 5.5, the protein wasdialysed in 30 mM Tris-HCl buffer, pH 7.9, using a centricon 3 KDacutoff membrane. α-Bungarotoxin and α-bungarotoxin¹²⁵I were a kind giftfrom Dr. Shalla Verrall.

Reagents and Fine Chemicals

DL-α-Lipoic acid, L-α-phosphatidylcholine from soybean, NADPH andβ-mercaptoethanol were purchased from Sigma Chemical Co. (St Louis, Mo.)and monobromobimane (mBBr, trade name thiolite) from Calbiochem (SanDiego, Calif.). Reagents for sodium dodecylsulfate (SDS)-polyacrylamidegel electrophoresis were purchased from Bio-Rad Laboratories (Richmond,Calif.).

Proteins and Enzymes

Thioredoxin and NTR were purified from E. coli as is described by Jiao,et al., (1992) Ag. Food Chem. (in press). Thioredoxin h was purifiedfrom wheat germ (Florencio, F. J., et al. (1988) Arch Biochem. Biophys.266:496-507) and thioredoxins f and m from spinach leaves (Florencio, F.J., et al., supra.). Human thioredoxin was a kind gift of Dr. EmanuelleWollman. NADP-malate dehydrogenase was purified from corn leaves(Jacquot, J.-P., et al. (1981) Plant Physiol. 68:300-304) andglutathione reductase from spinach leaves (Florencio, F. J. et al.,supra.). E. coli glutaredoxin was a kind gift of Professor A. Holmgren.

SDS-Polyacrylamide Gel Electrophoresis

SDS-polyacrylamide gel electrophoresis was performed in 10-20% gradientgels of 1.5 mm thickness that were developed for 3 hr at a constantcurrent of 40 mA. Following electrophoresis, gels were soaked for 2 hrin 12% (w/v) trichloroacetic acid and then transferred to a solutioncontaining 40% methanol and 10% acetic acid for 12 hr to remove excessmBBr. The fluorescence of protein-bound mBBr was determined by placinggels on a light box fitted with an ultraviolet light source (365 nm)Gels were photographed with Polaroid positive/negative Landfilm, type55, through a yellow Wratten gelatin filter No. 8 (cutoff=460 nm)(exposure time 40 sec. at f4.5). Gels were stained for protein for 1 hrin solution of 0.125% (w/v) Coomassie blue R-250 in 10% acetic acid and40% methanol. Gels were destained in this same solution from whichCoomassie blue was omitted.

Polaroid negatives of fluorescent gels and dry stained gels were scannedwith a laser densitometer (Pharmacia-LKB Ultroscan XL). The bands werequantified by evaluating areas or height of the peaks with Gelscan XLsoftware.

EXAMPLE 27 Reduction of Toxins and Labeling with mBBr

Reactions were carried out with 10 μg of target toxin in a final volumeof 100 μl in 30 mM Tris-HCl buffer, pH 7.9, with 0.8 μg thioredoxin, 0.7μg NTR (both from E. coli) and 2.5 mM NADPH. When thioredoxin wasreduced by DTT, NADPH and NTR were omitted and DTT was added to 0.5 mM.Assays with GSH were performed similarly, but at a final concentrationof 1 mM. For reduction by glutaredoxin, the thioredoxin and NTR werereplaced by 1 μg E. coli glutaredoxin, 0.38 μg glutathione reductase(partially purified from spinach leaves), 1 mM GSH and 2.5 mM NADPH (thecombination of these four components is called NADP/glutaredoxinsystem). Reduction by the reduced form of lipoic acid, was carried outin a volume of 100 μl at two concentrations, 100 μM and 200 μM, bothalone and with 0.8 μg of thioredoxin. The mixture was incubated for 2 hrat 37° C. in the case of erabutoxin b and α-bungarotoxin, 1 hr at roomtemperature for β-bungarotoxin and 20 min at room temperature forphospholipase A₂. After incubation, mBBr was added to 1.5 mM and thereaction continued for 15 min at room temperature. The reaction wasstopped and excess mBBr derivatized by adding 10 μl of 100 mMβ-mercaptoethanol, 5 μl of 20% SDS and 10 μl 50% glycerol. Samples werethen analyzed by SDS-polyacrylamide gel electrophoresis.

Total toxin reduction was accomplished by boiling samples for 3 min in 2mM DTT. After cooling, the samples were labeled with mBBr and treated asbefore, except that all samples were again boiled for 2 min prior toloading in the gel. The extent of the reduction of erabutoxin b by thedifferent reductants described above is shown in FIG. 49. Dithiothreitol(DTT) and the reduced forms of thioredoxin and lipoic acid are dithiolreductants as opposed to monothiol reductants like 2-mercaptoethanol andglutathione. DTT is a synthetically prepared chemical agent, whereasthioredoxin and lipoic acid occur within the cell. Evidence presentedabove demonstrates that lipoic acid is a more specific reductant thandithiothreitol. Dithiothreitol reduced the toxin partly withoutthioredoxin (lane 5) whereas reduced lipoic acid did not (lane 8). FIG.52 shows that the NTS or DTT plus thioredoxin are specific reductantsfor α-bungarotoxin and β-bungarotoxin.

EXAMPLE 28 NADP-Malate Dehydrogenase Activation

The ability of snake toxins to activate chloroplast NADP-malatedehydrogenase was carried out by preincubating 5 μg toxin with alimiting thioredoxin concentration (to restrict activation of the enzymeby the thioredoxin): E. coli thioredoxin, 0.25 μg; human, 0.9 μg; wheat,1.15 μg; spinach f and m, 0.375 and 0.125 μg, respectively. Purifiedcorn NADP-malate dehydrogenase, 1.4 μg, was added to a solutioncontaining 100 mM Tris-HCl, pH 7.9, thioredoxin as indicated, and 10 mMDTT (final volume 0.2 ml). After 25 min, 160 μl of the preincubationmixture was injected into a 1 cm cuvette of 1 ml capacity containing (in0.79 ml) 100 mM Tris HCl, pH 7.9, and 0.25 mM NADPH. The reaction wasstarted by the addition of 50 μl of 50 mM oxalacetic acid. NADPHoxidation was followed by monitoring the change in absorbance at 340 nmwith a Beckman spectrophotometer fitted with a four-way channel changer.FIG. 50 which represents the results of this experiment shows that thereduction by different reduced thioredoxins of erabutoxin bsignificantly alters the toxin's biological ability to activateNADP-malate dehydrogenase. The results demonstrate that, although thereare differences in effectiveness, all thioredoxins tested function tosome extent in limiting the effect of the toxin.

EXAMPLE 29 Proteolysis Assay of Erabutoxin b

Erabutoxin b, 10 μg was incubated for 2 hr at 37° C. with 30 mM Tris-HClbuffer pH 7.9 (total volume, 100 μl). As indicated, the buffer wassupplemented with 0.8 μg thioredoxin, 0.7 μg NTR and 2.5 mM NADPH. Whenthioredoxin was reduced by DTT the NTR and NADPH were omitted and DTTwas added to 0.5 mM. Following incubation, samples were digested with0.4 and 2 μg of trypsin for 10 min at 37° C. DTT, 4.8 μl of 50 mMsolution, 5 μl of 20% SDS and 10 μl of 50% glycerol were added, sampleswere boiled for 3 min, and then subjected to SDS-polyacrylamide gelelectrophoresis. Gels were stained with Coomassie blue and the proteinbands quantified by densitometric scanning as described above. Theresults of the assay are shown in Table XVI below.

These results show that reduction of a snake neurotoxin (erabutoxin b)renders the toxin more susceptible to proteolysis. An extension of thisconclusion would indicate that administration of reduced thioredoxin asa toxin antidote should help to destroy the toxin owing to the increasein proteolytic inactivation by proteases of the venom.

TABLE XVI Susceptibility of the Oxidized and Reduced Forms of Erabutoxinb to Trypsin % Erabutoxin b digested Treatment 0.4 μg trypsin 2 μgtrypsin Control  0.0 34.1 Reduced, NTS 21.1 57.8 Reduced, DTT  3.1 40.6Reduced, DTT + Trx 28.0 71.8 Erabutoxin b, 10 μg was preincubated for 2hours at 37° C. in 30 mM Tris-HCl buffer, pH 7.9, as follows: control,no addition; reduced by E. coli NADP/thioredoxin system (NTS),thioredoxin, NTR and NADPH; reduced by DTT, DTT; and reduced by DTT plusthioredoxin, DTT supplemented with E. coli thioredoxin. Afterpreincubation 0.4 μg and 2 μg of trypsin were added to the indicatedwhich then were analyzed by SDS-polyacrylamide gel electrophoresis.

EXAMPLE 30 Phospholipase A₂ Assay

Activity of the oxidized and reduced forms of the phospholipase A₂component of β-bungarotoxin was determined spectrophotometricallyfollowing change in acidity as described by Lobo de Araujo, et al.(1987) Toxicon 25:1181-1188. For reduction experiments, 10 μg toxin wasincubated in 30 mM Tris-HCl buffer, pH 7.9, containing 0.8 μgthioredoxin, 0.7 μg NTR and 7 mM NADPH (final volume, 35 μl). After 1 hrincubation at room temperature, 20 μl of the reaction mixture was addedto a 1 cm cuvette containing 1 ml of assay solution (adjusted to pH 7.6)that contained 10 mM CaCl₂, 100 mM NaCl, 4 mM sodium cholate, 175 μMsoybean phosphatidylcholine and 55 μM phenol red. The reaction wasfollowed by measuring the change in the absorbance at 558 nm in aBeckman Du model 2400 spectrophotometer. The results of this experimentwhich are shown in FIG. 51, demonstrate that β-bungarotoxin loses mostof its phospholipase activity when reduced by thioredoxin. The resultsare consistent with the conclusion that the administration of reducedthioredoxin following a snake bite would help detoxify the toxin byeliminating phospholipase A₂ activity.

EXAMPLE 31 α-Bungarotoxin Binding to Acetylcholine Receptor

α-Bungarotoxin binding was assayed with cultured mouse cells by usingradiolabeled toxin (Gu, Y., et al. (1985) J. Neurosci. 5:1909-1916).Mouse cells, line C₂, were grown as described by Gu et al (Gu, Y. etal., supra.) and plated in 24-well plastic tissue culture plates(Falcon) at a density of about 3000 cells per well. Growth medium wasreplaced by fusion medium after 48 hr and again after 96 hr. Cultureswere used for assay after an additional 2 days growth.

α-Bungarotoxin binding was determined with cells subjected to threedifferent treatments: [A] 10 nM α-bungarotoxin¹²⁵I (262 Ci/mmole) waspreincubated 2 hr at 37° C. in 200 μl of phosphate-saline buffer (0.15MNaCl in 10 mM Na₂HPO₄/NaH₂PO₄ pH 7.2) with 4 μg thioredoxin, 3.5 μg NTR(both from E. coli) and 6.25 mM NADPH. In certain cases, the NTR andNADPH were replaced by 1.25 mM DTT. After 2 hr incubation, the mixturewas transferred to a well containing mouse cells, washed two times withphosphate-saline, and incubated for 2 hours at 37° C. [B] After washingthe cells two times with phosphate-saline buffer, 10 nMα-bungarotoxin¹²⁵I (in 200 μl of phosphate-saline) was added per well.Following a 2 hr incubation at 37° C., cells were washed again withphosphate-saline buffer to remove unbound toxin. As indicated, 200 μlsaline, supplemented with 0.68 mM CaCl₂, 0.49 mM MgCl₂, 4 μgthioredoxin, 3.5 μg NTR and 6.25 mM NADPH were added to the well. Theplate was incubated 2 hr at 37° C. NTR and NADPH were omitted fromtreatment with DTT which was added at 1.25 mM. [C] After washing cellstwice with phosphate-saline buffer, 200 μl of a solution containing 4 μgthioredoxin, 3.5 μg NTR and 6.25 mM NADPH, were added to each well. Insome cases, NTR and NADPH were replaced with 1.25 mM DTT. The plate wasincubated for 2 hr at 37° C. Cells were then washed twice withphosphate-saline buffer to remove the added reductant. Phosphate-salinebuffer, 200 μl, containing 0.68 mM CaCl₂ and 0.49 mM MgCl₂ and 10 nMα-bungarotoxin¹²⁵I was added to each well. Incubation was continued for2 hr at 37° C. The results of this assay are shown in Table XVII. Thisexperiment shows that when reduced by thioredoxin, β-bungarotoxin can nolonger bind to the acetylcholine receptor. When extended to the wholeanimal, the thioredoxin-linked reduction mechanism would result indetoxification by eliminating binding of the toxin to its targetreceptor.

Each α-bungarotoxin binding assay was done in triplicate. Nonspecificbinding was measured by adding 100-fold excess unlabeled α-bungarotoxinto the incubation mixture. After the incubation period, the cells in allcases were washed with phosphate-saline to remove unbound toxin. Theamount of toxin bound was determined by solubilizing the cells in 0.1 MNaOH and measuring radioactivity in a gamma counter.

TABLE XVII Binding of α-Bungarotoxin to the Acetylcholine Receptor ofMouse Cells % Binding Treatment A

No reductant 100.0 NTS 0.0 DTT plus Thioredoxin 0.0 NTS minus NTR 63.0NTS minus Thioredoxin 78.0 NTS minus NADPH 101.0 Treatment B

No reductant 100.0 NTS 78.0 DTT plus Thioredoxin 76.0 Treatment C

No reductant 100.0 NTS 68.7 DTT 85.0 DTT plus Thioredoxin 68.8 E. coliNTS: thioredoxin, NTR and NADPH

EXAMPLE 32 Example for Detoxification in an Animal

Detoxification of snake neurotoxins is determined by subcutaneousinjection into mice. Assays are done in triplicate. Prior to injection,the toxin is diluted in phosphate-saline buffer (0.15M NaCl in 10 mMNa₂HPO₄/NaH₂PO₄ pH 7.2) at concentrations ranging up to twice the LD₅₀dose. (LD₅₀ is defined as that dose of toxin that kills 50% of a givengroup of animals.) For toxicity tests, the following neurotoxinconcentrations correspond to the LD₅₀ (per g mouse): erabutoxin b, 0.05μg-0.15 μg; α-bungarotoxin, 0.3 μg; and β-bungarotoxin, 0.089 μg. At 5,10, 30, 60 minutes and 4, 12 and 24 hr after injection, separate groupsof the challenged mice are injected (1) intravenously, and (2)subcutaneously (multiple local injections around the initial injectionsite). The thioredoxin is reduced with: (1) the E. coli NADP-thioredoxinsystem, using 0.08 μg thioredoxin, 0.07 μg NTR and 25 nanomoles NADPH;(2) Thioredoxin plus 1-2 nanomoles of reduced lipoic acid, using 0.08 μgE. coli or 0.20 μg human thioredoxin, and (3) using 0.08 μg E. coli or0.20 μg human thioredoxin with 5 nanomoles dithiothreitol(concentrations are per μg toxin injected into the animal; all solutionsare prepared in phosphate-saline buffer).

The effect of thioredoxin on detoxification is determined by (1)comparing the LD₅₀ with the control group without thioredoxin; (2)following the extent of the local reaction, as evidenced by necrosis,swelling and general discomfort to the animal; (3) following the serumlevels of creatin kinase, an indicator of tissue damage. Creatin kinase,which is released into the blood as a result of breakage of musclecells, is monitored using the standard assay kit obtained from SigmaChemical Co. (St. Louis, Mo.).

The symptoms of snake bite are multiple and depend on a variety offactors. As a consequence, they vary from patient to patient. There are,nonetheless, common symptoms that thioredoxin treatment should alleviatein humans. Specifically, the thioredoxin treatment should alleviatesymptoms associated with neurotoxic and related effects resulting fromsnake bite. Included are a decrease in swelling and edema, pain andblistering surrounding the bite; restoration of normal pulse rate;restriction of necrosis in the bite area; minimization of the affectedpart. A minimization of these symptoms should in turn result inimprovement in the general health and state of the patient.

CONCLUDING REMARKS

It can be seen from the foregoing general description of the inventionand from the specific examples illustrating applications thereof, thatthe invention has manifold and far reaching consequences. The inventionbasically provides novel dough and dough mixtures and novel methods forcreating new doughs and for improving the quality of dough and bakedgoods as well as novel methods for inactivating enzyme inhibitors incereal products. The invention also provides a novel method for alteringthe biological activity and inactivity of animal toxins, namely bee,scorpion and snake toxins. The invention further provides a novelprotein that is a pullulanase inhibitor and a method for itsinactivation.

While the invention has described in connection with certain specificembodiments thereof, it should be realized that various modifications asmay be apparent to those of skill in the art to which the inventionpertains also fall within the scope of the invention as defined by theappended claims.

What is claimed is:
 1. A method of reducing an amylase inhibitor proteinselected from the group consisting of CM and DSG proteins, said methodcomprising (a) adding a thiol redox protein selected from the groupconsisting of thioredoxin and glutaredoxin to a liquid or substancecontaining said amylase inhibitor, (b) reducing said thiol redoxprotein; and (c) reducing said inhibitor protein by said reduced thiolredox protein.
 2. The method of claim 1 wherein the thiol redox proteinis thioredoxin.
 3. The method of claim 2 wherein the thioredoxin isreduced by NADP-thioredoxin reductase in combination with NADPH.
 4. Themethod of claim 1 wherein the thiol redox protein is glutaredoxin. 5.The method of claim 1 wherein the amylase inhibitor protein is theα-amylase inhibitor CM-1, DSG-1 or DSG-2.
 6. A composition comprising anamylase inhibitor selected from the group consisting of a CM and DSGprotein, a thioredoxin, NADP-thioredoxin reductase and NADPH or an NADPHgenerating system.
 7. A method for inactivating an enzyme inhibitorcystine containing protein in a food product said inhibitor proteinrequiring intact disulfide bonds to inhibit said enzyme, said methodcomprising (a) mixing said food product with a thiol redox proteinselected from the group consisting of thioredoxin and glutaredoxin, (b)reducing said thiol redox protein, and (c) reducing said enzymeinhibitor by said reduced thiol redox protein, said reduction of saidinhibitor causing said inhibitor to be inactivated.
 8. The method ofclaim 1 wherein said food product is a cereal and said inhibitor is anamylase/subtilisin (asi) inhibitor, a CM protein or a DSG protein.